Reactive Red F03-0318

Endpoint summary

Description of key information

No classification for acute effects is required.

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:

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.

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 (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: RjHan:WI

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 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.

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.

Preliminary study:

Not applicable.

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:

other: 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 t

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

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

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.

 

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

GLP study conducted in accordance with OECD, EU and US guidelines therefore Klimisch 1 study.

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:

23 February 2011 to 9 March 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to EU, US & OECD test guidance in compliance with GLP and reported with a valid GLP certificate.

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

yes

Remarks:

the humidity (30 – 70%) was recorded out of the target range. The actual range was at the humidity 24-55 %.

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

yes

Remarks:

the humidity (30 – 70%) was recorded out of the target range. The actual range was at the humidity 24-55 %.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Deviations:

yes

Remarks:

the humidity (30 – 70%) was recorded out of the target range. The actual range was at the humidity 24-55 %.

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: RjHan:(WI) Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: RjHan:(WI) Wistar rats
Source: Laboratoire Elevage Janvier, B.P. 4105, Route des Chênes Secs, 53940 Le Genest-St-Isle CEDEX FRANCE
Hygienic level at arrival: SPF
Hygienic level during the study: Standard housing conditions
Justification of strain: The Wistar rat is one of the standard rodent species used in acute toxicity studies
Number of animals: 5 animals/sex
Sex: Male and female, female rats were nulliparous and non-pregnant.
Age of animals at study start: Young adult rats
Body weight range at dosing: Between 205 g and 240 g
Acclimatization time: 6 days

Husbandry
Animal health: Only healthy animals were used for the study. The veterinarian certified the health status.
Room-Box: 245/5
Housing: Individual caging
Cage type: Type II. polypropylene/polycarbonate
Bedding: Laboratory bedding: Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, 73494 Rosenberg, Germany);
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.4- 23.8 °C
Relative humidity: 24 - 55 %
Ventilation: 15-20 air exchanges/hour
Enrichment: Rodents were housed with deep wood sawdust bedding to allow digging and other normal rodent activities.

The temperature and relative humidity was 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 not considered 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.

Identification

The individual identification was performed using numbers written on the tail with a 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 containing information about study code, sex, dose group, cage number and individual animal numbers.

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: approximately 10 % area of the total body surface
- % coverage: approximately 10 % area of the total body surface)
- Type of wrap if used: semi occlusive plastic wrap

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period, the area of skin treated with the test item was washed with water of body temperature.
- Time after start of exposure: 14 days

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- Concentration (if solution): Not applicable
- Constant volume or concentration used: no. The appropriate amount of the test item was moistened with distilled water and distributed as uniformly as possible.

Duration of exposure:

14 days

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

no

Details on study design:

DOSAGES

Justification of the doses:
The test item was not expected to be lethal at 2000 mg/kg bw. A limit test was therefore performed.

PROCEDURE

The back of each animal was shaved (approximately 10 % area of the total body surface) approximately 24 hours prior to treatment. The test item was applied as a single dose as supplied to the shaved skin and remained in contact with the skin for the 24- hour exposure period. For that purpose, the appropriate amount of the test item was moistened with distilled water and distributed as uniformly as possible. Sterile gauze pads were placed on the skin of rats to cover the test item. These gauze pads were kept in contact with the skin by a patch with adhesive hypoallergenic plaster. The entire trunk of the animal was then wrapped with semi occlusive plastic wrap for 24 hours.

At the end of the exposure period, the area of skin treated with the test item was washed with water of body temperature.

OBSERVATIONS

CLINICAL OBSERVATIONS

Clinical observations were performed on the day of treatment at 1 and 5 hours after application of the test item and once each day for 14 days thereafter. Observations included the skin and fur, eyes and mucous membranes, the 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.

MEASUREMENT OF BODY WEIGHT

The body weights were recorded on Day 0 (before test item administration) and on Days 7 and 14.

NECROPSY

All animals were anaesthetised with Euthasol®40% (details in 3.1.3.) and exsanguinated. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed. All macroscopic changes were recorded.

EVALUATION

Body weight and body weight gain are summarized in tabular form. Clinical signs and necropsy findings are described and summarized in tabular form.

Statistics:

None

Preliminary study:

Not applicable

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred after a 24-hour dermal exposure to Reactive Red F03-0318 administered at 2000 mg/kg bw to RjHan:(WI) Wistar rats followed by a 14-day observation period.

Clinical signs:

other: No clinical signs were observed after the treatment with the test item or during the 14 day observation period.

Gross pathology:

At necropsy, no macroscopic test item-related findings were observed.

Other findings:

Red staining was recorded on the skin in all animals after dosing. The discoloration of the skin lasted up to Day 12 in all animals. No other local dermal signs were observed.

CLINICAL OBSERVATIONS

Dose level: 2000 mg/kg bw

Sex: Female

Cage No.

Animal No.

Observations

Observation days

Frequency

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

1h

5 h

1

4903

Symptom Free

+

+

+

+

+

5/16

Dark discoloration, red

-

-

+

+

+

+

+

+

+

+

+

+

+

-

-

-

1 1/16

2

4904

Symptom Free

+

+

+

+

+

5/16

Dark discoloration, red

-

-

+

+

+

+

+

+

+

+

+

+

+

-

-

-

1 1/16

3

4905

Symptom Free

+

+

+

+

4/16

Dark discoloration, red

-

-

+

+

+

+

+

+

+

+

+

+

+

+

-

-

12/16

4

4906

Symptom Free

+

+

+

+

+

5/16

Dark discoloration, red

-

-

+

+

+

+

+

+

+

+

+

+

+

-

-

-

1 1/16

5

4907

Symptom Free

+

+

+

+

4/16

Dark discoloration, red

-

-

+

+

+

+

+

+

+

+

+

+

+

+

-

-

12/16

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The acute dermal median lethal dose (LD50) of the test item Reactive Red F03 0318 was found to be higher than 2000 mg/kg body weight in male and female RjHan:(WI) Wistar rats.

Executive summary:

An acute dermal toxicity study was performed with test item Reactive Red F03-inRjHan:(WI) Wistar rats, in compliance with OECD Guideline No.: 402.

A limit test was carried out at 2000 mg/kg body weight (bw) in both sexes (5 rats/sex). The test item was applied as supplied, moistened with distilled water, as a single dermal 24-hour exposure followed by a 14‑day observation period. Clinical observations were performed on all animals at 1 and 5 hours after dosing and daily for 14 days thereafter. Body weight was measured prior to dosing on Day 0 and on Days 3, 7 and 14. Rats were euthanized and a gross macroscopic examination performed at the end of the 2-week observation period (Day 14).

The results of the study were summarized as follows:

 

Mortality

No mortality occurred.

 

Systemic clinical signs

No clinical signs were observed after the treatment with the test item or during the 14‑day observation period.

 

Local dermal signs

No local dermal signs were observed during the entire study period. However, red staining was observed on the skin in all animals after dosing from Day 1 to Day 12.

Body weight

The body weight and body weight gain of Reactive Red F03-0318 treated animals did not show any test item-related effect.

 

Necropsy

There was no evidence of test item-related observations at a dose level of 2000 mg/kg bw at necropsy. Uterus in estrus was observed in two females.

Conclusions

 

The acute dermal median lethal dose (LD₅₀) of the test item Reactive Red F03‑0318 was found to be higher than 2000 mg/kg bw in male and female RjHan:(WI) Wistar rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

GLP study conducted in accordance with OECD, EU and US guidelines therefore Klimisch 1 study.

Additional information

Testing on the above endpoints gave the following results:

 Acute toxicity: Oral.

-         LD50: >2000 mg/kg

Acute toxicity: Dermal.

-         LD50: >2000 mg/kg

Acute toxicity: Inhalation.

Not measured.

Justification for selection of acute toxicity – oral endpoint
Only 1 study available.

Justification for selection of acute toxicity – inhalation endpoint
The test substance has a predicted very low vapour pressure and is a granular product, hence the potential for the generation of inhalable forms is low. In addition the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be unlikely to occur. Dermal exposure is considered to be the appropriate route of exposure and has been assessed accordingly. No acute inhalation test was performed.

Justification for selection of acute toxicity – dermal endpoint
Only 1 study available.

Justification for classification or non-classification

The above studies have all been ranked reliability 1 according to the Klimisch et al system. This ranking was deemed appropriate because the studies were conducted to GLP and in compliance with agreed protocols. Sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds.

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for acute effects is therefore required.