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Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 October 2010 to 10 November 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to EU, OECD & US EPA 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
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
yes
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
yes
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
yes
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
yes

Test material

Constituent 1
Details on test material:
Name: Reactive Orange F08-0314

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system 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, 9-10 weeks old
Date of receipt: 21 October 2010
Body weight at treatment: 207 – 221 g
Acclimation period: At least 5 days
Animal health: Only healthy animals were used for the test. The veterinarian certified the health status.
Number of animal room: 522/7
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.
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.
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
Details on oral exposure:
Formulation: Test item was freshly formulated at a concentration of 200 mg/mL in the vehicle (without correction for purity), in the Central Dispensary Unit of LAB Research Ltd. on the day of administration. The formulation container was stirred continuously during administration to ensure that the syringe was filled from a homogenous liquid.

Doses
Justification of the 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.

Initially, three female animals were treated with 2000 mg/kg bw of Reactive Orange F08 0314. 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.
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
Three
Control animals:
no
Details on study design:
Procedure
A single oral dose was administered by gavage followed by a fourteen-day observation period. 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.

Clinical Observations
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.

Body Weight Measurement
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
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.
Statistics:
The method used was not intended to allow the calculation of a precise LD50 value.
The test item was ranked into categories of Globally Harmonized Classification System (GHS) described in the OECD Guideline No. 423.
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 Orange F08-0314 did not cause mortality at a dose level of 2000 mg/kg bw.
Clinical signs:
Treatment with Reactive Orange F08-0314 caused coloured urine and faeces at 3 hours after treatment in Group 1 and at 6 hours after treatment in Group 2.
Body weight:
Body weight gains of Reactive Orange F08-0314 treated animals during the study showed no indication of a treatment-related effect.
Gross pathology:
A single oral gavage of Reactive Orange F08-0314 to the RjHan:WI rat at a dose level of 2000 mg/kg bw followed by a 14 day observation period did not result in any test item related macroscopic findings.
Other findings:
No data.

Any other information on results incl. tables

CLINICAL OBSERVATIONS

DOSE LEVEL: 2000 mg/kg bw                                                                                                                          SEX: FEMALE

Cage No.

Animal Number

Observation

Observation days

Frequency

0

1-14

30’

1h

2h

3h

4h

6h

1*

2129

No clinical signs

+

+

+

+

+

+

+

20/20

2130

No clinical signs

+

+

+

+

+

+

+

20/20

2131

No clinical signs

+

+

+

+

+

+

+

20/20

DOSE LEVEL: 2000 mg/kg bw                                                                                                                                                     SEX: FEMALE

Cage No.

Animal Number

Observation

Observation days

Frequency

0

1-14

30’

1h

2h

3h

4h

6h

2**

2132

No clinical signs

+

+

+

+

+

+

+

20/20

2133

No clinical signs

+

+

+

+

+

+

+

20/20

2134

No clinical signs

+

+

+

+

+

+

+

20/20

Remarks:            +: present          -: absent

                               h=hours (s)        Treatment day = Day 0

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

                               *: Urine and faeces coloured (orange) in bedding at 3h after treatment

                               **: Urine and faeces coloured (orange) in bedding at 6h after treatment

 

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

1

2129

233

221

255

271

293

-12

34

16

22

60

2130

222

215

233

243

261

-7

18

10

18

39

2131

219

207

233

239

264

-12

26

6

25

45

Mean:

224.7

214.3

240.3

251.0

272.7

-10.3

26.0

10.7

21.7

48.0

Standard deviation:

7.4

7.0

12.7

17.4

17.7

2.9

8.0

5.0

3.5

10.8

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

2

2132

214

207

223

231

243

-7

16

8

12

29

2133

219

208

224

235

256

-11

16

11

21

37

2134

220

208

230

244

256

-12

22

14

12

36

Mean:

217.7

207.7

225.7

236.7

251.7

-10.0

18.0

11.0

15.0

34.0

Standard deviation:

3.2

0.6

3.8

6.7

7.5

2.6

3.5

3.0

5.2

4.4

Remark:              Treatment day = Day 0

 

NECROPSY FINDINGS

DOSE LEVEL: 2000 mg/kg bw                                                                                                                       SEX: FEMALE

Cage No.

Animal ID

External Observations

Internal Observations

Organ/Tissue

1

2129

No external observations recorded

No internal observations recorded

Not applicable

2130

No external observations recorded

No internal observations recorded

Not applicable

2131

No external observations recorded

No internal observations recorded

Not applicable

DOSE LEVEL: 2000 mg/kg bw                                                                                                                SEX: FEMALE

2

2132

No external observations recorded

No internal observations recorded

Not applicable

2133

No external observations recorded

No internal observations recorded

Not applicable

2134

No external observations recorded

No internal observations recorded

Not applicable

 

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 Orange F08-0314 was found to be above 2000 mg/kg bw in female RjHan: WI rats.

According the GHS criteria, Reactive Orange F08-0314 should be ranked as "Category 5" or "Unclassified".
Executive summary:

The single-dose oral toxicity of Reactive Orange F08-0314 was performed according to the acute toxic class method (OECD 423, OPPTS 870.1100, and Commission Regulation (EC) No 440/2008,B.1.tris) in RjHan: WI rats. The study is compliant to the Principles of Good Laboratory Practice (GLP) and reported with a valid GLP certificate.

 

Two groups of three female RjHan:WI rats (9-10 weeks old) were treated with Reactive Orange F08-0314 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 observed in 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 according to OECD 423 and Commission Regulation (EC) No 440/2008, B.1.tris.

 

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 Orange F08-0314 was administered as a solution prepared in distilled water at 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 Orange F08-0314 did not cause mortality at a dose level of 2000 mg/kg bw.

Clinical observations: Treatment with Reactive Orange F08-0314 caused coloured urine and faeces at 3 hours after treatment in Group 1 and at 6 hours after treatment in Group 2.

Body weight and body weight gain: Body weight gains of Reactive Orange F08-0314 treated animals showed no indication of a treatment-related effect.

Macroscopic Findings: A single oral gavage of 2000 mg/kg bw did not result in any test item related macroscopic findings.

 

Conclusion:

Under the conditions of this study, the acute oral LD50 value of the test item Reactive Orange F08-0314 was found to be above 2000 mg/kg bw in female RjHan: WI rats.

 

According the GHS criteria,Reactive Orange F08-0314should be ranked as "Category 5" or "Unclassified".