Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to OECD and GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

EC Number:

276-538-7

EC Name:

Tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Cas Number:

72252-58-5

Molecular formula:

C23H8Cr2N6O16S3.4Na

IUPAC Name:

tetrasodium [μ-[3-[[2-amino-5-hydroxy-6-[(2-hydroxy-5-nitro-3-sulphophenyl)azo]-7-sulpho-1-naphthyl]azo]-2-hydroxy-5-sulphobenzoato(8-)]]dichromate(4-)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Geniron Biolabs Pvt. Ltd., No.93, Solur, Thally Road, Anekal, Bengaluru - 562106, India

- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: Males : 229.4 to 234.3 g; Females: 215.3 to 225.8 g

- Housing: Animals were housed individually in standard polysulfone cages (Size: approximately L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottle. Additionally, polycarbonate rat huts were placed inside the cage as an enrichment object and were changed along with the cage at once a week.
Bedding: Steam sterilized corn cob was used and changed once a week along with the cage.

- Diet (e.g. ad libitum): Hypro rat & mice pellet feed, manufactured by Pranav Agro Industries Ltd., Pune 411 030, Maharashtra, India, was provided to animals
- Water (e.g. ad libitum): Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier manufactured by Eureka Forbes Ltd, Mumbai 400 001, India, was provided to animals in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: After physical examination for good health and suitability for experiment, the rats were acclimatized for six days before treatment under standard laboratory conditions. Animals were observed once daily during acclimatization period. Females were nulliparous and non-pregnant

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 23°C
- Humidity (%): 65 to 67 %
- Air changes (per hr): 14.1 to 14.3 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From : 29 September 2016 To : 19 October 2016

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

other: milli-Q water

Details on dermal exposure:

TEST SITE
- Area of exposure: Approximately 24 hours before treatment, the hair on the dorsolateral thoracic surface of the skin was clipped (approximately 10 x 8 cm) with an electric clipper (Aesculap- Germany).

Based on the individual body weight, the finely ground test item at the dose of 2000 mg/kg body weight was weighed on an aluminium foil and made into a paste by adding sufficient volume of Milli-Q water (approximately 1.4 mL) and completely transferred on to the cotton gauze (size: Males: 9 x 6 cm; Females: 8 x 5 cm of 6 ply) and applied (semi-occlusive) directly to the clipped skin of the rat to cover about 10% of body surface of the rat. Then the cotton was secured in position by adhesive tape wound around the torso. The test item contact period with the skin was for 24 hours.


REMOVAL OF TEST SUBSTANCE
- Washing (if done): After the 24 hour contact period, the dressing was removed and the applied area was washed with water and wiped dry using a clean towel.
- Time after start of exposure:10:27 AM to 10:34 AM

TEST MATERIAL

- Amount(s) applied (volume or weight with unit):
Refer Appendix 1

- Constant volume or concentration used: no
- For solids, paste formed: yes

VEHICLE
- Amount(s) applied (volume or weight with unit): Approxmately 1.4 mL

Duration of exposure:

24 hours

Doses:

limit dose: 2000 mg/kg

No. of animals per sex per dose:

5/sex/dose

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed for clinical signs and pre-terminal deaths (mortality) four times (at hourly intervals after application) during day 1 and twice daily on day 2 and 3 and once daily during Days 4 to 15. Individual body weights of animals were recorded on test days 1 (Pre-application), 8 (7 days post application), and 15 (14 days post application).
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, gross pathology.

Statistics:

The statistical analysis was not performed as the test was completed with the limit dose.

Results and discussion

Preliminary study:

No priliminary study

Effect levels

Sex:

male/female

Dose descriptor:

approximate LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality

Clinical signs:

other: There were no clinical signs observed during the study. However, black colour stain was observed on test item applied area in all rats on day 2 and 3 post dose observation

Gross pathology:

No abnormality was detected at necropsy

Any other information on results incl. tables

Table 1. Individual body weight, body weight changes and pre-terminal deaths

Group and Dose (mg/kg body weight)	Rat No.	S e x	Body weight (g)					No.dead / No.tested	Pre- terminal deaths (%)
			Initial (at treatment)	8^th   day	Weight change (day 8 – Initial)	15^th day	Weight change (day 15 – Initial)
G1 and 2000 Limit test	Rm5211	M	234.3	251.6	17.3	264.6	30.3	0/10	0
	Rm5212	M	230.8	246.8	16.0	258.9	28.1
	Rm5213	M	231.4	242.9	11.5	255.6	24.2
	Rm5214	M	229.4	245.1	15.7	257.3	27.9
	Rm5215	M	232.9	250.0	17.1	262.2	29.3
	Rm5216	F	220.8	232.6	11.8	245.6	24.8
	Rm5217	F	215.3	227.2	11.9	229.8	14.5
	Rm5218	F	223.8	234.8	11.0	245.1	21.3
	Rm5219	F	225.8	238.7	12.9	251.6	25.8
	Rm5220	F	218.9	231.3	12.4	243.4	24.5

F: Female             M: Male   

Table 2. Individual test item application, clinical signs and necropsy findings

Group and Dose (mg/kg body weight)

Date & Time of Application

Rat No.

Sex

Body weight initial (g)

Quantity  applied (mg)

Day of Observations

Day 1

2

3

1 hour

2 hours

3 hours

4 hours

AM

PM

AM

PM

G1 and 2000 Limit test

05 October 2016 and 10:27 AM to 10:34 AM

Rm5211

M

234.3

469

N

N

N

N

N*

N*

N*

N*

Rm5212

M

230.8

462

N

N

N

N

N*

N*

N*

N*

Rm5213

M

231.4

463

N

N

N

N

N*

N*

N*

N*

Rm5214

M

229.4

459

N

N

N

N

N*

N*

N*

N*

Rm5215

M

232.9

466

N

N

N

N

N*

N*

N*

N*

Rm5216

F

220.8

442

N

N

N

N

N*

N*

N*

N*

Rm5217

F

215.3

431

N

N

N

N

N*

N*

N*

N*

Rm5218

F

223.8

448

N

N

N

N

N*

N*

N*

N*

Rm5219

F

225.8

452

N

N

N

N

N*

N*

N*

N*

Rm5220

F

218.9

438

N

N

N

N

N*

N*

N*

N*

F: Female         M: Male          N:          AM: Ante Meridian        PM: Post    *:Black colour stain on test item applied area.

Table 2 contd. Individual test item application, clinical signs and necropsy findings

Group and Dose (mg/kg body weight)

Rat No.

Sex

Day of Observations

Necropsy Findings

4

5

6

7

8

9

10

11

12

13

14

15

G1 and 2000 Limit test

Rm5211

M

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5212

M

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5213

M

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5214

M

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5215

M

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5216

F

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5217

F

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5218

F

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5219

F

N

N

N

N

N

N

N

N

N

N

N

N

NAD

Rm5220

F

N

N

N

N

N

N

N

N

N

N

N

N

NAD

F: Female         M: Male          NAD: No Abnormality Detected                       N:          AM: Ante Meridian        PM: Post   

Applicant's summary and conclusion

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: not specified

Conclusions:

The acute dermal LD50 of the test item is more than 2000 mg/kg body weight in male and female Wistar rats.

Executive summary:

The acute dermal toxicity of the test item was tested in 5 male and 5 female Wistar rats with the limit dose of 2000 mg/kg body weight.

All the rats were observed for clinical signs of toxicity and mortality for 14 days post application. There were no clinical signs of toxicity and mortality. However, black colour stain was observed on test item applied area in all rats on day 2 and 3 post dose observation. All rats gained weight during experimental period. At the end of observation period, all surviving animals were euthanized and subjected to necropsy. There were no abnormalities detected at the necropsy.

Based on the present study results, the acute dermal LD₅₀of the test item is more than 2000 mg/kg body weight in male and female Wistar rats.