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Description of key information

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2500 mg/kg body weight.

The acute dermal LD50of the test item is more than 2000 mg/kg body weight in male and female Wistar rats.

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:
Betwen 26 May 2016 and 15 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
Female Wistar (RccHan™:WIST) strain rats were supplied by Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were 8 to 12 weeks of age. The body weight variation did not exceed ±20% of the mean body weight of any previously dosed animals.
Animal Care and Husbandry
The animals were housed in groups three in suspended solid floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately 3 to 4 hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
All animals were dosed once only by gavage
Doses:
Using available information on the toxicity of the test item, 2000 mg/kg was chosen as the starting dose.
Groups of fasted animals were treated as follows:
Dose Level(mg/kg) Concentration(mg/mL) Dose Volume(mL/kg) Number of Rats(Female)
2000 200 10 3
2000 200 10 3
No. of animals per sex per dose:
3 animals per dose, 2 dose groups.
Control animals:
no
Details on study design:
Using available information on the toxicity of the test item, 2000 mg/kg was chosen as the starting dose.
Groups of fasted animals were treated as follows:
Dose Level(mg/kg) Concentration(mg/mL) Dose Volume(mL/kg) Number of Rats(Female)
2000 200 10 3
2000 200 10 3
All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted body weight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each group to confirm the survival of the previously dosed animals.
The animals were observed for deaths or overt signs of toxicity 30 minutes, 1, 2 and 4 hours after dosing and subsequently once daily for 14 days.
Individual body weights were recorded prior to dosing and 7 and 14 days after treatment.
At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross pathological examination. This consisted of an external examination and opening of the abdominal and thoracic cavities for examination of major organs. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.
Statistics:
None recorded
Preliminary study:
Not applicable.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: 95% CL not reported
Mortality:
Individual mortality data are given in Table 1
There were no deaths
Clinical signs:
Individual clinical observations are given in Appendix 1.
Noisy respiration and/or hunched posture were noted in the first group of three treated animals. These animals appeared normal 4 hours or 2 days after dosing.
No signs of systemic toxicity were noted in the second group of three treated animals during the observation period.
Black staining of the feces and urine was noted in the cages of all animals.
Body weight:
Individual body weights and body weight change are given in Table2.
All animals showed expected gains in body weight over the observation period.
Gross pathology:
Individual necropsy findings are given in Table 3.
Black stained kidneys and stomach were noted at necropsy of the first group of three treated animals. No abnormalities were noted at necropsy of the second group of three treated animals.
Other findings:
None

Table1     Individual Clinical Observations and Mortality Data

Dose Level mg/kg

Animal Number and Sex

Effects Noted After Dosing
(Hours)

Effects Noted During Period After Dosing
(Days)

½

1

2

4

1UF

2U*F

3U*F

4U*F

5F·

6

7

8

9

10

11

12

13

14

2000

1-0

Female

H

H

H

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

1-1

Female

HRn

HRn

HRn

Rn

Rn

0

0

0

0

0

0

0

0

0

0

0

0

0

1-2

Female

HRn

HRn

HRn

Rn

Rn

0

0

0

0

0

0

0

0

0

0

0

0

0

2-0

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2-1

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

2-2

Female

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0


0=   No signs of systemic toxicity

H =  Hunched posture

Rn =Noisy respiration

F =  Black colored staining of the feces

F·= Black colored staining of the feces of animal numbers 1-0, 1-1 and 1-2

U =  Black staining of the urine

U* =Black staining of the urine in the cages of animal numbers 2-0, 2-1 and 2-2

Table 2     Individual Body Weights and Body Weight Changes

Dose Level
mg/kg

Animal Number
and Sex

Body Weight (g) at Day

Body Weight Gain (g) During Week

0

7

14

1

2

2000

1-0 Female

176

186

199

10

13

1-1 Female

173

184

192

11

8

1-2 Female

188

213

220

25

7

2-0 Female

184

194

217

10

23

2-1 Female

202

207

220

5

13

2-2 Female

198

205

221

7

16

Table 3     Individual Necropsy Findings

Dose Level
mg/kg

Animal Number
and Sex

Time of Death

Macroscopic Observations

2000

1-0 Female

Killed Day 14

Kidneys: stained black

Stomach: stained black

1-1 Female

Killed Day 14

Kidneys: stained black

Stomach: stained black

1-2 Female

Killed Day 14

Kidneys: stained black

Stomach: stained black

2-0 Female

Killed Day 14

No abnormalities detected

2-1 Female

Killed Day 14

No abnormalities detected

2-2 Female

Killed Day 14

No abnormalities detected

Interpretation of results:
GHS criteria not met
Conclusions:
The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2500 mg/kg body weight (Globally Harmonized Classification System - Unclassified).
Executive summary:

Introduction

The study was performed to assess the acute oral toxicity of the test item in the Wistar strain rat.

Methods

A group of three fasted females was treated with the test item at a dose level of 2000 mg/kg body weight. This was followed by a further group of three fasted females at the same dose level. Dosing was performed sequentially.

The test item was administered orally as a suspension in arachis oil BP. During the observation period of 14 days clinical signs and body weight development were monitored. Thereafter all animals were subjected to gross necropsy.

Results

Mortality. There were no deaths.

Clinical Observations. Noisy respiration and/or hunched posture were noted in the first group of three treated animals. There were no signs of systemic toxicity noted in the second group of three treated animals. Black staining of the feces and urine was noted in the cages of all animals.

Body Weight. All animals showed expected gains in body weight.

Necropsy. Black stained kidneys and stomach were noted at necropsy of the first group of three treated animals. No abnormalities were noted at necropsy of thesecond group of three treated animals.

Conclusion

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2500 mg/kg body weight (Globally Harmonized Classification System - Unclassified).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 500 mg/kg bw
Quality of whole database:
1 reliable without restriction

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD and GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Geniron Biolabs Pvt. Ltd., No.93, Solur, Thally Road, Anekal, Bengaluru - 562106, India

- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: Males : 229.4 to 234.3 g; Females: 215.3 to 225.8 g

- Housing: Animals were housed individually in standard polysulfone cages (Size: approximately L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottle. Additionally, polycarbonate rat huts were placed inside the cage as an enrichment object and were changed along with the cage at once a week.
Bedding: Steam sterilized corn cob was used and changed once a week along with the cage.

- Diet (e.g. ad libitum): Hypro rat & mice pellet feed, manufactured by Pranav Agro Industries Ltd., Pune 411 030, Maharashtra, India, was provided to animals
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd, Mumbai 400 001, India, was provided to animals in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: After physical examination for good health and suitability for experiment, the rats were acclimatized for six days before treatment under standard laboratory conditions. Animals were observed once daily during acclimatization period. Females were nulliparous and non-pregnant

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 23°C
- Humidity (%): 65 to 67 %
- Air changes (per hr): 14.1 to 14.3 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From : 29 September 2016 To : 19 October 2016
Type of coverage:
semiocclusive
Vehicle:
other: milli-Q water
Details on dermal exposure:
TEST SITE
- Area of exposure: Approximately 24 hours before treatment, the hair on the dorsolateral thoracic surface of the skin was clipped (approximately 10 x 8 cm) with an electric clipper (Aesculap- Germany).

Based on the individual body weight, the finely ground test item at the dose of 2000 mg/kg body weight was weighed on an aluminium foil and made into a paste by adding sufficient volume of Milli-Q water (approximately 1.4 mL) and completely transferred on to the cotton gauze (size: Males: 9 x 6 cm; Females: 8 x 5 cm of 6 ply) and applied (semi-occlusive) directly to the clipped skin of the rat to cover about 10% of body surface of the rat. Then the cotton was secured in position by adhesive tape wound around the torso. The test item contact period with the skin was for 24 hours.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the 24 hour contact period, the dressing was removed and the applied area was washed with water and wiped dry using a clean towel.
- Time after start of exposure:10:27 AM to 10:34 AM

TEST MATERIAL

- Amount(s) applied (volume or weight with unit):
Refer Appendix 1

- Constant volume or concentration used: no
- For solids, paste formed: yes

VEHICLE
- Amount(s) applied (volume or weight with unit): Approxmately 1.4 mL
Duration of exposure:
24 hours
Doses:
limit dose: 2000 mg/kg
No. of animals per sex per dose:
5/sex/dose
Control animals:
not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed for clinical signs and pre-terminal deaths (mortality) four times (at hourly intervals after application) during day 1 and twice daily on day 2 and 3 and once daily during Days 4 to 15. Individual body weights of animals were recorded on test days 1 (Pre-application), 8 (7 days post application), and 15 (14 days post application).
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, gross pathology.
Statistics:
The statistical analysis was not performed as the test was completed with the limit dose.
Preliminary study:
No priliminary study
Sex:
male/female
Dose descriptor:
approximate LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality
Clinical signs:
There were no clinical signs observed during the study. However, black colour stain was observed on test item applied area in all rats on day 2 and 3 post dose observation
Body weight:
All rats gained body weight throughout the observation period.
Gross pathology:
No abnormality was detected at necropsy

Table 1. Individual body weight, body weight changes and pre-terminal deaths

Group and

Dose

(mg/kg

body weight)

Rat

No.

S

e

x

Body weight (g)

No.dead /

No.tested

Pre- terminal deaths (%)

Initial

(at treatment)

8th  

day

Weight change

(day 8 – Initial)

15th

day

Weight change

(day 15 – Initial)

 

G1 and

2000

Limit test

 

Rm5211

M

234.3

251.6

17.3

264.6

30.3

 

0/10

 

0

Rm5212

M

230.8

246.8

16.0

258.9

28.1

Rm5213

M

231.4

242.9

11.5

255.6

24.2

Rm5214

M

229.4

245.1

15.7

257.3

27.9

Rm5215

M

232.9

250.0

17.1

262.2

29.3

Rm5216

F

220.8

232.6

11.8

245.6

24.8

Rm5217

F

215.3

227.2

11.9

229.8

14.5

Rm5218

F

223.8

234.8

11.0

245.1

21.3

Rm5219

F

225.8

238.7

12.9

251.6

25.8

Rm5220

F

218.9

231.3

12.4

243.4

24.5

F: Female             M: Male   

Table 2. Individual test item application, clinical signs and necropsy findings

Group and Dose

(mg/kg body weight)

Date &

Time of

Application

Rat

No.

Sex

Body weight initial

(g)

Quantity

 applied

(mg)

 

Day of Observations

Day 1

2

3

1

hour

2

hours

3

hours

4

hours

 

 

AM

PM

AM

PM

G1

and

2000

Limit test

05 October 2016

and

10:27 AM

to

10:34 AM

Rm5211

M

234.3

469

N

N

N

N

N*

N*

N*

N*

Rm5212

M

230.8

462

N

N

N

N

N*

N*

N*

N*

Rm5213

M

231.4

463

N

N

N

N

N*

N*

N*

N*

Rm5214

M

229.4

459

N

N

N

N

N*

N*

N*

N*

Rm5215

M

232.9

466

N

N

N

N

N*

N*

N*

N*

Rm5216

F

220.8

442

N

N

N

N

N*

N*

N*

N*

Rm5217

F

215.3

431

N

N

N

N

N*

N*

N*

N*

Rm5218

F

223.8

448

N

N

N

N

N*

N*

N*

N*

Rm5219

F

225.8

452

N

N

N

N

N*

N*

N*

N*

Rm5220

F

218.9

438

N

N

N

N

N*

N*

N*

N*

F: Female         M: Male          N:          AM: Ante Meridian        PM: Post    *:Black colour stain on test item applied area.

Table 2 contd. Individual test item application, clinical signs and necropsy findings

Group and Dose

(mg/kg body weight)

Rat

No.

Sex

Day of Observations

Necropsy

Findings

4

5

6

7

8

9

10

11

12

13

14

15

G1

and

2000

Limit test

Rm5211

M

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5212

M

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5213

M

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5214

M

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5215

M

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5216

F

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5217

F

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5218

F

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5219

F

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5220

F

N

N

N

N

N

N

N

N

N

N

N

N

NAD

F: Female         M: Male          NAD: No Abnormality Detected                       N:          AM: Ante Meridian        PM: Post   

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: not specified
Conclusions:
The acute dermal LD50 of the test item is more than 2000 mg/kg body weight in male and female Wistar rats.
Executive summary:

The acute dermal toxicity of the test item was tested in 5 male and 5 female Wistar rats with the limit dose of 2000 mg/kg body weight.

All the rats were observed for clinical signs of toxicity and mortality for 14 days post application. There were no clinical signs of toxicity and mortality. However, black colour stain was observed on test item applied area in all rats on day 2 and 3 post dose observation. All rats gained weight during experimental period. At the end of observation period, all surviving animals were euthanized and subjected to necropsy. There were no abnormalities detected at the necropsy.

Based on the present study results, the acute dermal LD50of the test item is more than 2000 mg/kg body weight in male and female Wistar rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw

Additional information

Justification for classification or non-classification

No classification

The criteria for classification are not met.

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