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Administrative data

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-06-12 to 2012-08-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
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 (incl. QA statement)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)
Test type:
standard acute method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(3-)
EC Number:
260-906-9
EC Name:
Trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(3-)
Cas Number:
57693-14-8
Molecular formula:
C40H20CrN6O14S2.3Na
IUPAC Name:
trisodium bis[3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(3-)
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. . 0715)
- Free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 261111)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
water
Remarks:
Aqua ad injectionem (Diprom, lot no. 10952-1, expiry date: 09/2013)
Details on dermal exposure:
Preparation of 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.

Application:
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.
The dressing consisted of a gauze-dressing and non-irritating tape and was fixed with an additional dressing in a suitable manner.
Duration of exposure:
The test item was held in contact with the skin throughout a 24-hour period.
At the end of the exposure period the residual test item was removed using aqua ad injectionem.
Doses:
The test item was applied at a single dose of 2000 mg/kg body weight to each animal considering the active component content of 86.5 %.
No. of animals per sex per dose:
5 male and 5 female
Control animals:
not required
Details on study design:
Observation period: all animals were observed for 14 days after dosing.

Weight assessment: 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; 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.

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
approximate LD50
Effect level:
> 2 000 mg/kg bw
Mortality:
No mortality was observed
Clinical signs:
No treatment-related effects were observed.
Body weight:
The body weight development of all male and female animals was within the expected range.
Gross pathology:
No treatment-related effects were observed.
Other findings:
No erythema or oedema was observed.

Any other information on results incl. tables

Clinical signs of systemic toxicity – Individual data - males:

Animal
No. / sex / dose

Time

Observations

21/ male /
2000 mg/kg bw

during the whole observation period

no signs of toxicity

22/ male /
2000 mg/kg bw

during the whole observation period

no signs of toxicity

23/ male /
2000 mg/kg bw

during the whole observation period

no signs of toxicity

24/ male /
2000 mg/kg bw

during the whole observation period

no signs of toxicity

25/ male /
2000 mg/kg bw

during the whole observation period

no signs of toxicity

Clinical signs of systemic toxicity – individual data – females:

Animal
No. / sex / dose

Time

Observations

26/ female / 2000 mg/kg bw

during the whole observation period

no signs of toxicity

27/ female / 2000 mg/kg bw

during the whole observation period

no signs of toxicity

28/ female / 2000 mg/kg bw

during the whole observation period

no signs of toxicity

29/ female / 2000 mg/kg bw

during the whole observation period

no signs of toxicity

30/ female / 2000 mg/kg bw

during the whole observation period

no signs of toxicity

Skin irritation – individual data – males:

Day after start of application

Animal No. 21

Animal No. 22

Animal No. 23

Animal No. 24

Animal No. 25

E/O

Comments

E/O

Comments

E/O

Comments

E/O

Comments

E/O

Comments

day 2

0/0

R

0/0

R

0/0

R

0/0

R

0/0

R

day 3

0/0

R

0/0

R

0/0

R

0/0

R

0/0

R

day 4

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 5

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 6

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 7

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 8

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 9

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 10

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 11

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 12

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 13

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 14

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 15

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

Comments:

E = erythema; O = oedema; 1, 2, 3, 4 = scoring system laid down in OECD guideline 404;

R = residues of test item

Skin irritation – individual data – females:

Day after start of application

Animal No. 26

Animal No. 27

Animal No. 28

Animal No. 29

Animal No. 30

E/O

Comments

E/O

Comments

E/O

Comments

E/O

Comments

E/O

Comments

day 2

0/0

R

0/0

R

0/0

R

0/0

R

0/0

R

day 3

0/0

R

0/0

R

0/0

R

0/0

R

0/0

R

day 4

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 5

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 6

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 7

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 8

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 9

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 10

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 11

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 12

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 13

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 14

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

day 15

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

0/0

nsf

Comments:

E = erythema; O = oedema; 1, 2, 3, 4 = scoring system laid down in OECD guideline 404;

R = residues of test item

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

250

271

306

22

22 / male

239

257

288

21

23 / male

228

243

270

18

24 / male

240

255

282

18

25 / male

254

277

303

19

26 / female

215

222

228

6

27 / female

208

216

229

10

28 / female

214

226

235

10

29 / female

218

242

250

15

30 / female

221

226

230

4

Macroscopic findings - individual data – males and females:

Dose: 2000 mg/kg bw

Animal no. /
sex

Organ

Macroscopic findings

21 / male

-

nsf

22 / male

-

nsf

23 / male

-

nsf

24 / male

-

nsf

25 / male

-

nsf

26 / female

-

nsf

27 / female

-

nsf

28 / female

-

nsf

29 / female

-

nsf

30 / female

-

nsf

nsf = no specific findings

LD50:

Dose (unit)

 

No. of animals investigated

No. 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

Applicant's summary and conclusion

Interpretation of results:
other: not classified according to the CLP Regulation (EC 1272/2008)
Conclusions:
LD50 > 2000 mg/kg bw.

Executive summary:

Method

Test substance was applied to  5 rats per sex at dose of 2000 mg/kg for a 24 h exposure duration.

Results

No death occurred.

Signs of toxicity related to dose level used, time of onset and duration: no treatment-related effects were observed.

Effect on organs (related to dose level): no treatment-related effects were observed.

Signs of irritation: no erythema or oedema was observed.

LD50 > 2000 mg/kg bw.