Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 February 2011 to 02 March 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU & OECD test guidance in compliance with GLP and reported with a valid GLP certificate.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: Reactive Red F03-0318

Test animals

Species:
rat
Strain:
other: RjHan:WI
Sex:
female
Details on test animals and environmental conditions:
Species and strain: RjHan:WI rats
Source: Laboratoire Elevage Janvier, B.P. 4105, Route des Chênes Secs, 53940 Le Genest-St-Isle CEDEX FRANCE
Hygienic level at supplier: SPF
Hygienic level during the study: Standard housing conditions
Number of animals: 6 animals, 3 animals/group
Sex: Female, nulliparous and non-pregnant.
Age of animals at dosing: Young healthy adult rats, ~ 9 weeks old
Date of receipt: 10 February 2011
Body weight at treatment: 222 – 248 g
Acclimation period: At least 5 days

Husbandry
Animal health: Only healthy animals were used for the test. The veterinarian certified the health status.
Number of animal room: 522/4
Housing: 3 animals / cage
Cage type: Type III polypropylene/polycarbonate
Bedding: Lignocel Bedding for Laboratory Animals was available to animals during the study.
Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: 15-20 air exchanges/hour
Enrichment: Animals were housed by group to allow social interaction and with deep wood sawdust bedding to allow digging and other normal rodent activities.

The temperature and relative humidity were recorded twice daily during the study.

Food and Water Supply
Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from the municipal supply, as for human consumption from 500 ml bottle ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water quality control analysis is performed once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). The quality control results are retained in the archives at LAB Research Ltd.

Animal Identification

Animals were individually identified using numbers written on the tail with an indelible marker pen. The numbers were given on the basis of LAB Research Ltd.' s Master File, for each animal allocated to the treatment groups. The cages were identified by cards, with information about study code, sex, dose group, cage number and individual animal numbers.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 200 mg/mL (without correction for purity)
- Amount of vehicle (if gavage): A constant treatment volume of 10 mL/kg bodyweight was applied.
- Justification for choice of vehicle: Not provided
- Lot/batch no. (if required): 7530810
- Purity: Not applicable
- Dose preparation: Test item was freshly formulated on the day of administration. The formulation container was stirred continuously during administration to ensure that the syringe was filled from a homogenous liquid.

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

- Rationale for the selection of the starting dose: The initial dose level was selected on the basis of the information provided by the Sponsor. The LD50 value was expected to be above 2000 mg/kg bw.

Procedure:
A single oral dose was administered by gavage. The animals were fasted for about 16 hours prior to treatment. The food but not water was withheld during an overnight period. Animals were weighed just before treatment. The test item was administered by oral gavage in the morning. The food was returned 3 hours after the treatment. A constant treatment volume of 10 mL/kg bodyweight was applied.
Doses:
Initially, three female animals were treated with 2000 mg/kg bw of Reactive Red F03 -0318. As no mortality occurred within 24 hours after dosing, a second group of three animals received 2000 mg/kg bw Reactive Orange F08 0314 approximately 24 hours after treatment of the first group. No mortality occurred in the second treatment group; hence, further testing was not required according to the test guidelines. The test was terminated on completion of the 14-day observation period.
No. of animals per sex per dose:
3 initial female + 3 additional female
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical observations were performed on all animals at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Individual observations were performed on the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. The body weight was recorded on the day before treatment (Day -1), on the day of the treatment (Day 0) and on study days 3, 7, and 14.
- Necropsy of survivors performed: Macroscopic examination was performed on all animals. The animals were sacrificed by exsanguination under pentobarbital anaesthesia (Euthasol® 40 %). After examination of the external appearance, the cranial, thoracic and the abdominal cavities were opened and the organs and the tissues were observed. Macroscopic abnormalities were recorded.
- Other examinations performed: None
Statistics:
Clinical signs, body weight, body weight gain and gross macroscopic data were tabulated.

Results and discussion

Preliminary study:
Not applicable.
Effect levels
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
Reactive Red F03-0318 did not cause mortality at a dose level of 2000 mg/kg bw.
Clinical signs:
Treatment with Reactive Red F03-0318 caused reddish coloured faeces in both treated groups on Day 0 and 1 after treatment, red-brown staining of the fur in Group 1 at 6 hours after the treatment, and liquid faeces in two animals of Group 2 on the day of treatment.
Body weight:
Body weight gains of Reactive Red F03-0318 treated animals during the study showed no indication of a treatment-related effect.
Gross pathology:
No test substance-related findings were noted at necropsy in Group 1 rats. In Group 2 animals, dark/pink diffuse discoloration of the skin and subcutis, kidneys, spleen, uterus, tail and/or mesenteric lymph nodes were noted at necropsy. These changes result most likely from the staining effect of the red dye and caused no adverse toxicological effects under the conditions of this study.
Other findings:
None

Any other information on results incl. tables

CLINICAL OBSERVATIONS

DOSE LEVEL: 2000 mg/kg bw

SEX: FEMALE

Cage No.

Animal Number

Observations

Observation days

Frequency

0

1

2-14

30'

1h

2h

3h

4h

6h

1 *

4655

Symptom Free

+

+

+

+

+

-

+

+

19/20

Red-brown staining (fur)

-

-

-

-

-

+

-

-

1/20

4656

Symptom Free

+

+

+

+

+

-

+

+

19/20

Red-brown staining (fur)

-

-

-

-

-

+

-

-

1/20

4657

Symptom Free

+

+

+

+

+

-

+

+

19/20

Red-brown staining (fur)

-

-

-

-

-

+

-

-

1/20

2 *

4658

Symptom Free

+

+

+

-

-

-

+

+

17/20

Faeces liquid

-

-

-

+

+

+

-

-

3/20

4659

Symptom Free

+

+

+

+

+

+

+

+

20/20

4660

Symptom Free

+

+

+

-

-

-

+

+

17/20

Faeces liquid

-

-

-

+

+

+

-

-

3/20

 Remarks:           +: present                                 -: absent

h=hour (s)                                Treatment day= Day 0

*: Red faeces on Day 0 and 1

Frequency of observation = number of occurence of observation / total number of observations

BODY WEIGHT DATA

DOSE LEVEL: 2000 mg/kg bw

SEX: FEMALE

Cage No.

Animal No.

Body weight (g) Days

Body Weight Gain (g)

 

-1

0

3

7

14

-1-0

0-3

3-7

7- 14

-1 - 14

 

4655

258

237

259

277

314

-21

22

18

37

56

1

4656

262

244

270

288

321

-18

26

18

33

59

 

4657

255

237

260

272

281

-18

23

12

9

26

 

4658

239

222

238

242

248

-17

16

4

6

9

2

4659

259

244

264

280

284

-15

20

16

4

25

 

4660

264

248

265

285

319

-16

17

20

34

55

Mean:

256.2

238.7

259.3

274.0

294.5

-17.5

20.7

14.7

20.5

38.3

Standard deviation:

9.0

9.2

11.2

16.7

28.8

2.1

3.8

5.9

15.7

21.0

Remark: Treatment day= Day 0

NECROPSY FINDINGS

DOSE LEVEL: 2000 mg/kg bw

SEX: FEMALE

Cage No.

Animal ID

Necropsy Date

External Observations

Internal Observations

Organ/Tissue

1

4655

01 March 2011

No external observations recorded

In estrus

Uterus

4656

01 March 2011

No external observations recorded

No internal observations recorded

Not applicable

4657

01 March 2011

No external observations recorded

Dark discoloration, red, diffuse, all lobes

Lungs

4658

02 March 2011

Tail: Dark discoloration, pink, diffuse

Dark discoloration, pink, diffuse, whole body

Skin & Subcutis

2

4659

02 March 2011

Tail: Dark discoloration, pink, diffuse

Dark discoloration, pink, diffuse, bilateral

Kidneys

Dark discoloration, pink, diffuse, whole body

Skin & Subcutis

4660

02 March 2011

Tail: Dark discoloration, pink, diffuse

Small

Spleen

Dark discoloration, pink, diffuse, whole body

Skin & Subcutis

Dark discoloration, pink, diffuse, horns, bilateral

Uterus

Dark discoloration, pink

Lymph node, mesenteric

Dark discoloration, pink, diffuse, bilateral

Kidneys

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, the acute oral LD50 value of the test item Reactive Red F03-0318 was found to be above 2000 mg/kg bw in female
RjHan: WI rats. According the EU criteria, Reactive Red F03-0318 should be ranked as not classified.
Executive summary:

The single-dose oral toxicity of Reactive Red F03-0318 was performed according to the acute toxic class method (OECD 423 and Commission Regulation (EC) No 440/2008,B.1.tris) in RjHan: WI rats.

Two groups of three female RjHan:WIrats (ca.9 weeks old) were treated with Reactive Red F03-0318 at a dose level of 2000 mg/kg bw (Group 1 and Group 2).

 

Initially, three females (Group 1) were treated at a dose level of 2000 mg/kg bw. Rats were maintained without compound administration for a 2‑week observation period after the day of dosing. As no mortality was observedin this dose group within 24 hours after dosing, a confirmatory treatment was performed on 3 further females (Group 2) at the same dose level. As no mortality was observed in the second dose group, no further treatment was needed.

 

A single oral treatment was carried out by gavage for each animal after an overnight food withdrawal (about 16 hours prior to treatment). Food was made available again 3 hours after the treatment. Reactive Red F03-0318 was administered as a solution prepared in distilled waterat a concentration of 200 mg/mL at a dosing volume of 10 mL/kg bw.

 

Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Body weight was measured on Days -1, 0, 3 and 7 and before necropsy. All animals were subjected to a necropsy and a macroscopic examination.

 

Results

Mortality

Reactive Red F03-0318 did not cause mortality at a dose level of 2000 mg/kg bw.

Clinical observations

Treatment with Reactive Red F03-0318 caused reddish coloured faeces in both treated groups on Day 0 and 1 after treatment, red-brown staining of the fur in Group 1 at 6 hours after the treatment, and liquid faeces in two animals of Group 2 on the day of treatment.

Body weight and body weight gain

Body weight gains of Reactive Red F03-0318 treated animals showed no indication of a treatment-related effect.

 

Macroscopic Findings

No test substance-related findings were noted at necropsy in Group 1 rats. In Group 2 animals, dark/pink diffuse discoloration of the skin and subcutis, kidneys, spleen, uterus, tail and/or mesenteric lymph nodes were noted at necropsy. These changes result most likely from the staining effect of the red dye and caused no adverse toxicological effects under the conditions of this study.

Conclusion:

Under the conditions of this study, the acute oral LD50value of the test item Reactive Red F03-0318 was found to be above 2000 mg/kg bw in female RjHan: WI rats. According the EU criteria, Reactive Red F03-0318 should be ranked as not classified.