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Description of key information

PB25

Under the conditions of the present study, a single oral application of the test item to female rats at a dose of 2000 mg/kg body weight was associated with no sign of toxicity or mortality. The LD50 for female rats is > 2000 mg/kg body weight

Under the conditions of the present study, single dermal application of the test item to rats at a dose of 2000 mg/kg body weight was associated with no mortality and neither signs of toxicity nor signs of irritation. The dermal LD50 was determined to be > 2000 mg/ kg body weight.

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:
From 06 MAR 2012 to 27 MAR 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 423) and according to GLP.
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
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
no
GLP compliance:
yes
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:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 150-174 g
- Fasting period before study: 16-19 h
- Housing: in groups in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 110811)
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 0815), ad libitum
- Water: tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/-10
- Air changes (per hr): 10 x
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
cotton seed oil
Details on oral exposure:
VEHICLE
- Amount of vehicle (if gavage): 10 mL / kg bw
- Justification for choice of vehicle: The vehivle was chosen due to its non-toxic characteristics
- Lot/batch no. (if required): MKBG0088V

MAXIMUM DOSE VOLUME APPLIED: 10 mL / kg bw

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: highest required dose tested.
Doses:
The starting dose was selected to be 2000 mg/kg bw. No compound-related mortality was recorded for any animal of step 1 or 2. Based on these results and according to the acute toxic class method regime no further testing was required.
No. of animals per sex per dose:
3 females per step / 2 steps performed
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were weighed on day 1 (prior to administratin) and on days 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight
Statistics:
no
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no animal died during the 14-day observation period
Mortality:
All animals survived until the end of the study.
Clinical signs:
None of the animals showed any sign of toxicity.
Body weight:
None of the animals showed weight loss during the observation period.
Gross pathology:
With the exception of acute injection of blood vessels in the abdominal region, which is due to the euthanasia injection, no specific gross pathological changes were recorded for any animal.

Table: Body Weight Development - Absolute Body Weights in g and Body Weight Gain in %

Animal No. /
Sex

g
Day 1

g
Day 8

g
Day 15

%
Day 1-15

Step 1 (2000 mg/kg Body Weight)

1 / female

166

192

193

16

2 / female

174

189

203

17

3 / female

150

167

170

13

Step 2 (2000 mg/kg Body Weight)

4 / female

156

179

184

18

5 / female

170

194

200

18

6 / female

166

183

194

17

Table: LD50 Cut-Off

Dose
(unit)

Number of
Animals
Investigated

Number of Intercurrent Deaths

LD50 Cut-Off

2000 mg/kg bw

6

0

unclassified

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, a single oral application of the test item to female rats at a dose of 2000 mg/kg body weight was associated with no sign of toxicity or mortality. The LD50 for female rats is > 2000 mg/kg body weight
Executive summary:

Two groups, each of three female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight. The test item was suspended in cotton seed oil at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg.

All animals used in the study were allowed to acclimatise to the laboratory conditions for at least 5 days. The animals were observed on delivery, on inclusion in the study and before administration for mortality/morbidity and other clinical signs. All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. Their body weights were recorded on day 1 (prior to the administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

Table1:  Results per Step

Step

Sex/No.

Dose (mg/kg)

Number of Animals

Number of Intercurrent Deaths

1

female/1-3

2000

3

0

2

female/4-6

2000

3

0

All animals survived until the end of the study without showing any signs of toxicity.

Throughout the 14-day observation period, the body weight gain of the test animals was within the normal range of variation for this strain.

At necropsy, no macroscopic findings were observed in any animal of any step. The LD50 for rats is > 2000 mg/kg body weight

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
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:
From 01 MAR 2012 to 04 MAY 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 402) and according to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1200 (Acute Dermal Toxicity)
Deviations:
no
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: Harlan Winkelmann, 33178 Borchen, Germany
- Age at study initiation: males: 9 to 10 weeks; females 12 to 13 weeks
- Weight at study initiation: males: 229 to 241 g; females: 211-224 g
- Housing: The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 110811)
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 1114), ad libitum
- Water: tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: Adequate acclimatisation period (at least five days) under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10 x / hour
- Photoperiod: 12 hours light, 12 hours dark
- Full barrier in an air-conditioned room
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
Type of coverage:
semiocclusive
Vehicle:
other: cottonseed oil
Details on dermal exposure:
Preparation of the Animals:
The animals were marked for individual identification by tail painting.
Approximately 24 hours before the test, the fur was removed from the dorsal area of the trunk using an electric clipper.
Care was taken to avoid abrading the skin, and only animals with healthy intact skin were used.
No less than 10% of the body surface was cleared for the application.
Prior to the application a detailed clinical observation was made of all animals.

TEST SITE
The test item was applied at a single dose, uniformly over an area which was approximately 10% of the total body surface.
The test item was held in contact with the skin by a dressing throughout a 24-hour period.
- Type of wrap if used: The dressing consisted of a gauze-dressing and non-irritating tape and was fixed with an additional dressing in a suitable manner

REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test item was removed using the vehicle cottonseed oil
- Time after start of exposure: 24 hours (i.e. at the end of the exposure period)

VEHICLE
- Justification: The vehicle was chosen due to its minimal potential influence on irritation of the skin.
- Lot/batch no. (if required): MKBG0088V (Sigma Aldrich, expiry date: 2012-05)
Duration of exposure:
24 hours period
Doses:
2000 mg/kg body weight
No. of animals per sex per dose:
5
Control animals:
not required
Details on study design:
Observation period:
All animals were observed for 14 days after dosing

Weight Assessment:
The animals were weighed on day 1 (prior to the application) and on days 8 and 15.

Clinical Examination:
careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes
and with special attention given during the first 4 hours post-dose). As soon as symptoms were noticed they were recorded.
Thereafter, the animals were observed for clinical signs once daily until the end of the observation period. All abnormalities were recorded.
Cageside observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory,
circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined.
Attention was directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Pathology:
At the end of the observation period the surviving animals were sacrificed with an overdosage of pentobarbital injected
intraperitoneally (Narcoren®, Merial) at the dosage of approximately 8 mL/kg bw. All animals were subjected to gross necropsy.
All gross pathological changes were recorded and in case of findings the tissues were preserved for a possible histopathological evaluation.
The preserved tissues of which no histopathological evaluation was made will be discarded 3 months after the release of the final report
unless otherwise agreed upon with the sponsor.

Evaluation of Results:
Individual reactions of each animal were recorded at each time of observation.
Toxic response data were recorded by sex and dose level.
Nature, severity and duration of clinical observations were described.
The body weight changes were summarised in a tabular form.
Necropsy findings were described.
Statistics:
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results,
a statistical evaluation of the results is not regarded as necessary.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no animal died during the 14-day observation period
Mortality:
No mortality was observed
Clinical signs:
No treatment-related effects were observed.






Body weight:
A slight weight loss was recorded for 2 out of 5 female animals during the first week, but all of the female animals showed
weight gain during the second week. The effects on weight development might be secondary to the dressing, and toxicological
relevance of this finding cannot clearly be concluded.
The male animals showed weight gain during the first and the second week of the observation.
Gross pathology:
No treatment-related effects were observed.
Other findings:
No erythema or oedema was observed.

Table Absolute Body Weights in g and Body Weight Gain in %:

Dose: 2000 mg/kg body weight

Animal No. / Sex

g
Day 1

g
Day 8

g
Day 15

%
Day 1-15

21 / male

233

257

279

20

22 / male

231

251

281

22

23 / male

234

240

262

12

24 / male

241

261

285

18

25 / male

229

237

289

26

26 / female

211

212

221

5

27 / female

217

222

223

3

28 / female

221

219

225

2

29 / female

219

225

232

6

30 / female

224

223

228

2

Table LD50:

Dose (Unit)

 

Number of Animals Investigated

Number of Intercurrent Deaths

LD50

2000 mg/kg bw

5 males

0

> 2000 mg/kg bw

2000mg/kg bw

5 females

0

> 2000 mg/kg bw

bw = body weight

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, single dermal application of the test item to rats at a dose of 2000 mg/kg body weight was associated with no mortality and neither signs of toxicity nor signs of irritation. The dermal LD50 was determined to be > 2000 mg/ kg body weight.
Executive summary:

To test acute dermal toxicity 5 male and 5 female Wistar rats were subjected to a test according to OECD test guideline 402. The test material was applied once in cottonseed oil at a dose of 2000 mg/kg bw for 24 hours under semi-occlusive conditions to the skin of rats. During the 14 -day observation period none of the tested animals died, showed specific signs of systemic toxicity or revealed signs of dermal irritation. No findings were made at the macroscopic examination performed at the end of the observation period. The rather low weight gain (2 -6 % during the observation period) was not accounted for as adverse as there were no effects seen at the clinical and pathological examinations. Study authors mentioned that they might be secondary to the dressing. Under the conditions of the present study the dermal LD50 was determined to be > 2000 mg/kg body weight.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Justification for classification or non-classification

No classification, as no adverse effects were observed

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