2-Naphthalenesulfonic acid, 7-amino-4-hydroxy-3-[[2-methoxy-5-methyl-4-[[2-(sulfooxy)ethyl]sulfonyl] phenyl]azo]-8-[[2-sulfo-4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]azo]-, tetrasodium salt

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Principles of method if other than guideline:

The objective of this acute dermal toxicity study was to assess the toxicological profile of the test item on application as a single semi-occlusive dermal application to rats.

GLP compliance:

yes

Test type:

fixed dose procedure

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 6-hydroxy-5-[(4-sulphonatophenyl)azo]naphthalene-2-sulphonate

EC Number:

220-491-7

EC Name:

Disodium 6-hydroxy-5-[(4-sulphonatophenyl)azo]naphthalene-2-sulphonate

Cas Number:

2783-94-0

Molecular formula:

C16H12N2O7S2.2Na

IUPAC Name:

disodium (5E)-6-oxo-5-[(4-sulfonatophenyl)hydrazinylidene]naphthalene-2-sulfonate

Details on test material:

- Name of test material: FD&C Yellow No. 6
- IUPAC name: disodium 6-hydroxy-5-[(4-sulfonatophenyl)diazenyl]naphthalene-2-sulfonate
- Molecular formula: C16H12N2O7S2.2Na
- Molecular weight: 454.38
- Substance type: organic
- Physical state: solid
- Purity: 91.8%

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Vivo Bio Tech Ltd. Telangana
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 8 to 10 Weeks
- Weight at study initiation: 159.10 g to 169.08 g
- Fasting period before study: Rats were fasted overnight
- Identification:By rat accession number. Identification of individual rats was by cage card and turmeric colour body markings. The rat accession number was allotted during the course of the study. The temporary body marking during acclimatization period was done with crystal violet.
- Housing:Rats 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 once a week
- Diet (e.g. ad libitum): Rat & Mice pellet feed, manufactured by Krishna Valley Agro Tech LLP, MIDC Kupwad block, Sangli, Maharashtra, 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 animals were acclimatized five days for G1-FTS and ten days for G1-STS before treatment. Animals were observed once daily during acclimatization period. Females were nulliparous and non-pregnant

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 59 to 67%,
- Air changes (per hr): air conditioned with adequate fresh air supply (12.4 air changes/hour)
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.

IN-LIFE DATES: From

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: dorsolateral thoracic surface of the skin
- % coverage: 10% of the body surface of the rat
- Type of wrap if used: The applied area was covered with cotton gauze

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The dressing was removed and the applied area was washed with deionized water and wiped dry using clean towel.
- Time after start of exposure:24 hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The undiluted test item at the doses of 200 (0.20 mL/kg body weight), 1000 (1 mL/kg body weight) and 2000 (1.99 mL/kg body weight) was applied

Duration of exposure:

24 hours

Doses:

DRF G1 - 2000 mg/kg
Main G2- 2000 mg/kg

No. of animals per sex per dose:

3 [ 1 female/group for dose range finding study and 2 female / main study group (1 for dose range finding study and 2 for main study)

Control animals:

not specified

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) once during first 30 minutes after application, and at hourly intervals for 6 hours after application on the day of treatment (day 1) and once daily during Days 2 to 15.
- Necropsy of survivors performed: Yes
- Other examinations performed: Clinical examination and pre-terminal deaths: All rats were observed for changes in skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern. Attention was directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
Body weights: Individual body weights of animals were recorded on test days 1 (Pre-application), 8 (7 days post application), and 15 (14 days post application).
Gross Pathology: At the end of the observation period, all rats were euthanised and exsanguinated under isoflurane anesthesia and subjected to detailed necropsy by an experienced prosector and the findings were recorded. Microscopic examination was not carried out as no gross pathological changes were observed.

Statistics:

not specified

Results and discussion

Preliminary study:

Dose range finding study - Selection of dose level: An initial starting dose of 200 mg/kg body weight was tested with 1 female rat (dose range finding study). As there was no mortality at this dose range finding study the dose range finding study was continued with 1 female rat (dose range finding study) at the dose of 1000 mg/kg body weight. There was no mortality, hence the dose range finding study was continued with 1 female rat (dose range finding study) at the dose of 2000 mg/kg body weight.

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was observed

Mortality:

There were no pre-terminal deaths (mortality) observed during the study.

Clinical signs:

other: There were no clinical signs observed during the study.

Gross pathology:

No abnormality was detected at necropsy.

Other findings:

not specified

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)					Pre-terminal deaths
			Initial (Day 1 - at treatment)	8^th   day	Weight change (day 8 – Initial)	15^th day	Weight change (day 15 – Initial)
G1 and 2000 DRF	Rm8779	F	229.80	244.45	14.65	232.63	2.83	0
G1 and 2000 Main study	Rm8780	F	237.85	239.22	1.37	248.70	10.85	0
	Rm8781	F	240.16	242.81	2.65	249.49	9.33	0

 DRF: Dose Range Finding   F: Female

 

APPENDIX 2.     contd. Individual test item application, clinical signs, skin reaction and necropsy findings

Dose range finding study

Group & Dose (mg/kg body weight)

Date and time of application

Rat Number

S e x

Initial Bwt (g)

Quantity (mg) applied

Observations and skin reaction

Days

1

2

3

4

5

30  min

1 h

2 h

3 h

4 h

5 h

6 h

*

Er @

Ed @

*

Er @

Ed @

*

Er @

Ed @

G1 and 2000 DRF

05 April 2018 and 10.27 AM

Rm8779

F

229.80

460

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

 

Group & Dose (mg/kg body weight)

Animal Number

S e x

Observation

Necropsy findings

Days

6

7

8

9

10

11

12

13

14

15

G1 and 2000 DRF

Rm8779

F

N

N

N

N

N

N

N

N

N

N

NAD

F: Female             N: Normal          h: hour    min: minutes                      NAD: No abnormality detected      Er: Erythema                      Ed: Edema  

Score 0: No Erythema / Edema       

*: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

APPENDIX 2 contd. Individual test item application, clinical signs, skin reaction and necropsy findings

 

main study

 

Group & Dose (mg/kg body weight)

Date and time of application

Rat Number

S e x

Initial Bwt (g)

Quantity (mg) applied

Observations and skin reaction

Days

1

2

3

4

5

30 min

1 h

2 h

3 h

4 h

5 h

6 h

*

Er @

Ed @

*

Er @

Ed @

*

Er @

Ed @

G1 and 2000 Limit test Main

10 April 2018 and 10.14AM and 10.15 AM

Rm8780

F

237.85

476

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

Rm8781

F

240.16

480

N

N

N

N

N

N

N

N

N

0

0

N

0

0

N

0

0

 

Group & Dose (mg/kg body weight)

Rat Number

S e x

Observations

Necropsy findings

Days

6

7

8

9

10

11

12

13

14

15

G2 and 2000 Limit test Main

Rm8780

F

N

N

N

N

N

N

N

N

N

N

NAD

Rm8781

F

N

N

N

N

N

N

N

N

N

N

NAD

F: Female             N: Normal          h: hour    min: minutes                       NAD: No abnormality detected      Er: Erythema                       Ed: Edema  

Score 0: No Erythema / Edema       

    

*: Clinical signs; @: Skin scoring as per Draize method (approximately 24, 48 and 72 hours) after test patch removal

 

  

Applicant's summary and conclusion

Interpretation of results:

other: Not classified

Conclusions:

It was concluded that the acute dermal median lethal dose (LD50) of the given test chemical, when administered to female Wistar rats was considered to be >2000 mg/kg body weight. Thus, according to CLP criteria for acute toxicity rating for the chemicals, it infers that the given test chemical does not classify as an acute dermal toxicant. CLP Classification: “Not classified”.

Executive summary:

The acute dermal toxicity of the given test chemical was tested with 2000 mg/kg with 1 female for the dose range finding study, followed by additional 2 females for main study at the dose of 2000 mg/kg body weight in Wistar rats. Based on the individual body weight, the test item at the dose of 2000 mg/kg body weight was weighed on an aluminum foil and made as a paste in Milli-Q water and applied directly to the clipped skin (Clipping was done approximately 24 hour prior to treatment) of the animal to cover about 10% of the body surface of the animal (semi-occlusive). The area of application was covered with cotton gauze (size: Females: 8 x 5 cm of 6 ply) and it was secured in position by adhesive tape wrapped around the torso. The test item contact period with the skin was for 24 hours.After the 24 hours contact period, the dressing was removed and the applied area was washed with deionized water and wiped dry using clean towels. All the rats were observed for clinical signs of toxicity and mortality for 14 days post application. In addition, the treatment site was observed at 24, 48 and 72 hours after removal of test item using the Draize criteria. There were no clinical signs of toxicity and mortality. There was no skin reaction observed at test item applied area. Body weight was measured on days 1, 8 and 15 and 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 given test chemical is more than 2000 mg/kg body weight in female Wistar rats.