Disodium 2-(2,4,5,7-tetrabromo-6-oxido-3-oxoxanthen-9-yl)benzoate

Endpoint summary

Description of key information

Acute oral toxicity: 

The acute oral toxicity dose (LD50) was considered based on different studies conducted on rats and mice for the test chemical.The study concluded that the LD50 value is >2000 mg/kg bw, for acute oral toxicity.Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute oral toxicity. 

Acute Inhalation Toxicity:

The acute inhalation toxicity study need not be conducted because exposure to humans via inhalation route is not likely taking into account due to the low vapour pressure of the test chemical, which is reported to be 2.54E-020 mm Hg = 3.39E-018 Pa at 25 deg C. Thus, exposure to inhalable dust, mist and vapour of the chemical is highly unlikely. Therefore this study is considered for waiver. 

 

Acute Dermal toxicity:

The acute dermal toxicity dose (LD50) was considered based on experimental study conducted on rats for the test chemical. The study concluded that the LD50 value is >2000 mg/kg bw, for acute dermal toxicity. Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute dermal toxicity.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from experimental study report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Principles of method if other than guideline:

The objective of this acute oral toxicity study was to assess the toxicological profile of the test item when administered to rats by a single oral gavage.

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Geniron Biolabs Pvt. Ltd, Bengaluru
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 to 12 Weeks
- Weight at study initiation: 169.70 g to 189.74 g
- 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.
- Fasting period before study: rats were fasted for approximately 17 hours.
- Housing:Rats were housed individually in standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill
- Diet (e.g. ad libitum): Rat & Mice pellet feed, ad libitum
- 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, Mumbai, ad libitum
- Acclimation period: The animals were acclimatized six days for G1-FTS and eleven days for G1-STS before treatment. Animals were observed once daily during acclimatization period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 24°C
- Humidity (%): 56 to 67%
- Air changes (per hr): air conditioned with adequate fresh air supply (12.9 air changes/hour)
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.

IN-LIFE DATES: From: 22 June 2018 To: 31 August 2018

Route of administration:

oral: gavage

Vehicle:

other: Milli-Q water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 2000 mg/kg body weight (G1 First and second treatment steps)
- Amount of vehicle (if gavage):10 mL/kg

DOSAGE PREPARATION (if unusual): A required quantity of test item was weighed in beaker and small volume of the Milli-Q water was added and mixed by using glass rod and transferred to a measuring cylinder. The beaker was rinsed with vehicle and all the rinsing was quantitatively transferred into the measuring cylinder. The final volume was made up to the mark with Milli-Q water to get the desired test item concentration of 200 mg/mL. The prepared dose formulation was transferred to the dedicated labeled beaker. Preparations were made prior to dosing.

Doses:

G1 (FTS) - 2000 mg/kg
G1 (STS) - 2000 mg/kg

No. of animals per sex per dose:

G1 (FTS) - 2000 mg/kg - 3
G1 (STS) - 2000 mg/kg - 3

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical signs and pre-terminal deaths - At each step, the animals were observed five times on test day 1 (day of administration) i.e. at 30 minutes and four times at hourly intervals and once daily during days 2 to 15 post administration. Observations included 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 the observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma and all observed clinical signs were recorded.

- Body weights - The body weights were recorded on test day 1 (pre-administration), day 8 (7 days post administration) and day 15 (14 days post administration).

- Necropsy of survivors performed: yes, the rats surviving to the end of the observation period were euthanised by using isoflurane anaesthesia and subjected to detailed necropsy. Gross
pathological findings were recorded and reported. Microscopic examination was not carried out as no gross pathological changes were observed.

Statistics:

not specified

Preliminary study:

not specified

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

G1 - [2000 mg/kg body weight - Treatment (FTS and STS)]: There were no pre-terminal deaths were observed.

Clinical signs:

other: G1 - [2000 mg/kg body weight - Treatment (FTS and STS)]: There were no clinical signs observed in any of the rats, except reddish brown faeces on days 2 and 3 of the observation period which is due to the colour of the test item.

Gross pathology:

There were no gross pathological changes at necropsy.

Other findings:

not specified

TABLE 1.   Body weight, body weight change and pre-terminal deaths

 

Group and

Body weight (g)

No. dead/

Pre-

Dose

Rat

Weight

Weight

Day of Death

terminal

(mg/kg

No.

Sex

Initial

th

change

th

change

At

(Time of Death)

No.

deaths

body weight)

(Day 1)

8 day

(day 8 –

15 day

(day 15

Death

tested

(%)

Initial)

– Initial)

G1

Rw211

F

176.39

181.69

5.3

186.17

9.78

NA

NA

(FTS)

Rw212

F

178.59

181.85

3.26

187.22

8.63

NA

NA

0/3

0

2000

Rw213

F

183.89

195.05

11.16

199.81

15.92

NA

NA

G1

Rw214

F

176.99

184.00

7.01

187.32

10.33

NA

NA

(STS)

Rw215

F

169.70

171.39

1.69

180.53

10.83

NA

NA

0/3

0

2000

Rw216

F

189.74

196.18

6.44

197.84

8.1

NA

NA

F: Female

FTS: First Treatment Step

STS: Second Treatment Step

NA: Not Applicable   0: No deaths

 

Interpretation of results:

other: Not classified

Conclusions:

Based on the results of the present study, the LD50 value of the given test chemical is >2000 mg/kg bw. Thus, the test item is not classified for acute oral toxicity. CLP criteria "Not Classified".

Executive summary:

The acute oral toxicity study was conducted to assess the toxicological profile of the test item as per OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method) in Wistar rats.

The dose formulation was prepared by using Milli-Q water and administered as a single oral gavage to overnight fasted (approximately 17 hours) three female rats (G1-FTS) at the dose of 2000 mg/kg body weight. There were no clinical signs of toxicity and pre-terminal deaths.

Hence, a confirmatory test was done at 2000 mg/kg body weight (G1-STS) with three additional female rats as per Annex 2d of the guideline OECD 423. There were no clinical signs of toxicity and pre-terminal deaths. Based on the scheme - Annex 2d of the guideline OECD 423, the dose was stopped.

The rats were observed for mortality and clinical signs for 14 days post treatment. Body weights were recorded prior to dosing on day 1 and on days 8 and 15. Necropsy was performed for all the rats at termination. All survived rats gained weight during experimental period. There were no gross pathological changes at necropsy.

Based on the results of the present study, the LD50 value of the given test chemical is >2000 mg/kg bw. Thus, the test item is not classified for acute oral toxicity. CLP criteria "Not Classified".

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Data is Klimisch 1 and from study report.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Quality of whole database:

Waiver

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report.

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:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Geniron Biolabs Pvt. Ltd., Bengaluru
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 to 12 Weeks
- Weight at study initiation: Females: 217.20 to 222.36 g
- Identification:By rat accession number. Identification of individual rats is by cage card and crystal violet colour body markings. The temporary body marking during acclimatization period was done with crystal violet. The rat accession numbers were allotted during the course of the study and was included in raw data and reported.
- Housing: Animals were housed individually in standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill. Additionally, polycarbonate rat huts were placed inside the cage as enrichment objects and were changed along with the cage once a week. Bedding: Steam sterilized corn cob was used and changed once a week along with the cage.
- Diet (e.g. ad libitum): Rat & Mice pellet feed, ad libitum
- 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, ad libitum
- Acclimation period: The animals were acclimatized six days for G1-DRF, eleven days for G2-DRF, thirteen days G3-DRF and fifteen days for G3-Main before treatment. Animals were observed once daily during acclimatization period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 24°C
- Humidity (%): 56 to 67 %
- Air changes (per hr): air conditioned with adequate fresh air supply (12.9 air changes/hour).
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 22 June 2018 To: 31 August 2018

Type of coverage:

semiocclusive

Vehicle:

other: Milli-Q water

Details on dermal exposure:

TEST SITE
- Area of exposure: the hair on the dorsolateral thoracic surface of the skin was clipped (approximately 10 x 8 cm) with an electric clipper.
- % coverage: about 10% of body surface of the rat.
- Type of wrap if used: The cotton gauze was secured in position by adhesive tape wound around the torso.

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
- For solids, paste formed: yes, the test item at dose of 200 (G1-DRF), 1000 (G2-DRF) and 2000 (G3-DRF and Main) mg/kg body weight, was weighed on an aluminium foil and made into a paste by adding sufficient volume (about 0.1 mL, 0.2 mL, 0.3 mL and 0.4 mL) of Milli-Q water.

Duration of exposure:

24 hours

Doses:

Three treatment group
G1 – DRF
G2 – DRF
G3 – DRF and G3-Main

No. of animals per sex per dose:

G1 – DRF - 1
G2 – DRF - 1
G3 – DRF and G3-Main - 3

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical examination and pre-terminal deaths - 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. In addition, treatment site at was observed 24, 48 and 72 hours after removal of test item using the Draize criteria (Refer Annexure 4 of this report). 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).

- Necropsy of survivors performed: yes, 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

Preliminary study:

Dose range finding study - As there was no acute dermal toxicity data available, an initial dose of 200 mg/kg body weight was tested in 1 female rat (dose range finding study) followed by 1000 mg/kg and 2000 mg/kg (dose range finding study). Based on the outcome of these dose range-finding studies, the main study was conducted with 2 further animals to confirm the classification. There was no test item-related mortality. The subsequent dosing was done approximately 2-4 days after the previous dosing.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

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

TABLE 1.	Individual body weight, body weight changes and pre-terminal deaths
Group and				S				Body weight (g)				Pre-terminal
Dose		Rat										deaths
						Initial
				e			8th	Weight change		15th	Weight change
(mg/kg body weight)		No.		x		(Day 1 - at	day	(day 8 – Initial)		day	(day 15 – Initial)
						treatment)
G1		Rw223
200				F		221.66	223.87	2.21		225.29	3.63	0
DRF
G2		Rw224
1000				F		217.20	220.14	2.94		222.61	5.41	0
DRF
G3		Rw225
2000				F		221.00	223.12	2.12		226.92	5.92	0
DRF
G3		Rw226		F		219.14	220.89	1.75		224.83	5.69	0
2000
Main study		Rw227		F		222.36	225.16	2.8		230.45	8.09	0
DRF: Dose Range		Finding	F: Female

 

 

Interpretation of results:

other: Not classified

Conclusions:

Based on the present study results, the acute dermal LD50 of the given test chemical is >2000 mg/kg body weight in female Wistar rats. The test item does not classified for acute dermal toxicity.
CLP classification "Not classified”.

Executive summary:

The acute dermal toxicity study was conducted as per OECD Guideline 402 (Acute Dermal Toxicity) for the given test chemical tested with 200 mg/kg, 1000 mg/kg and 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 200 mg/kg, 1000 mg/kg and 2000 mg/kg body weight was weighed on an aluminium foil and made into a paste by adding sufficient volume of the Milli-Q water and completely transferred on to the cotton gauze (size: Females: 8 x 5 cm of 6 ply) and applied directly (semi-occlusive) to the clipped skin of the rat to cover about 10% of body surface of the rat. It was secured in position by adhesive tape wound 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. 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 LD50 of the given test chemical is >2000 mg/kg body weight in female Wistar rats. The test item does not classified for acute dermal toxicity. CLP classification "Not classified”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Data is Klimisch 1 and from study report.

Additional information

Acute oral toxicity:

In different studies, the given test chemical has been investigated for acute oral toxicity to a greater or lesser extent. Often are the studies based on in-vivo experiments in rodents, i.e. most commonly in rats and mice for test chemical. The studies are summarized as below:

 

The acute oral toxicity study was conducted to assess the toxicological profile of the test item as per OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method) in Wistar rats.

The dose formulation was prepared by using Milli-Q water and administered as a single oral gavage to overnight fasted (approximately 17 hours) three female rats (G1-FTS) at the dose of 2000 mg/kg body weight. There were no clinical signs of toxicity and pre-terminal deaths.

Hence, a confirmatory test was done at 2000 mg/kg body weight (G1-STS) with three additional female rats as per Annex 2d of the guideline OECD 423. There were no clinical signs of toxicity and pre-terminal deaths. Based on the scheme - Annex 2d of the guideline OECD 423, the dose was stopped.

The rats were observed for mortality and clinical signs for 14 days post treatment. Body weights were recorded prior to dosing on day 1 and on days 8 and 15. Necropsy was performed for all the rats at termination. All survived rats gained weight during experimental period. There were no gross pathological changes at necropsy.

Based on the results of the present study, the LD50 value of the given test chemical is >2000 mg/kg bw. Thus, the test item is not classified for acute oral toxicity. CLP criteria "Not classified".

 

The above study is supported with another study mentioned in different handbooks, authoritative databases and secondary reports for the test chemical. In an acute oral toxicity study, mice were treated with the given test chemical at the dose concentration of 2344 mg/kg bw orally. Animals were observed for mortality. 50% mortality was observed in treated mice at 2344 mg/kg bw. Therefore, the LD50 value was considered to be 2344 mg/kg bw, when mice were treated with the given test chemical orally.

 

Both the above studies are contradicted with the study mentioned in secondary source for the test chemical. In an acute oral toxicity study, rats were treated with the given test chemical at the dose concentration of 1000 mg/kg bw orally. Animals were observed for mortality. No mortality was observed in treated rats at 1000 mg/kg bw. Therefore, the LD50 value was considered to be >1000 mg/kg bw, when rats were treated with test chemical orally.

 

Thus, based on the above summarised experimental studies on test chemical, the LD50 value is >2000 mg/kg bw. Therefore, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute oral toxicity.

 

Acute Inhalation Toxicity:

The acute inhalation toxicity study need not be conducted because exposure to humans via inhalation route is not likely taking into account due to the low vapour pressure of the test chemical, which is reported to be 2.54E-020 mm Hg = 3.39E-018 Pa at 25 deg C. Thus, exposure to inhalable dust, mist and vapour of the chemical is highly unlikely. Therefore this study is considered for waiver. 

 

Acute Dermal Toxicity:

The acute dermal toxicity study was conducted as per OECD Guideline 402 (Acute Dermal Toxicity) for the given test chemical tested with 200 mg/kg, 1000 mg/kg and 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 200 mg/kg, 1000 mg/kg and 2000 mg/kg body weight was weighed on an aluminium foil and made into a paste by adding sufficient volume of the Milli-Q water and completely transferred on to the cotton gauze (size: Females: 8 x 5 cm of 6 ply) and applied directly (semi-occlusive) to the clipped skin of the rat to cover about 10% of body surface of the rat. It was secured in position by adhesive tape wound 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 deionised water and wiped dry using clean towels.

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. 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 LD50 of the given test chemical is >2000 mg/kg body weight in female Wistar rats. The test item does not classify for acute dermal toxicity. CLP classification "Not classified”.

 

Justification for classification or non-classification

Based on the above studies on test chemical, it can be concluded that the LD50 value is >2000 mg/kg bw, for acute oral and acute dermal toxicity. Thus, comparing this value with the criteria of CLP regulation, the given test chemical cannot be classified for acute oral and acute dermal toxicity. For acute inhalation toxicity wavier was added so, not possible to classify.