3-nitrobenzoic acid

Endpoint summary

Description of key information

Acute oral toxicity: 

Acute oral toxicity dose (LD50) was considered based on experimental study conducted on rats for the given test chemical. The LD50 value was considered is >2000 mg/kg bw. 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 toxicity inhalation study need not be conducted because exposure to humans via inhalation route is not likely taking into account the low vapour pressure of the substance, which is reported as 0.0000371 mm Hg at 25°C. Thus, exposure to inhalable dust, mist and vapour of the test 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 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:

no

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: 9 to 11 Weeks
- Weight at study initiation: 142.08 g to 167.18 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 16 to 18 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 five days for G1-FTS, ten days for G1-STS, thirteen days for G2-FTS and sixteen days for G2-STS. Animals were observed once daily during acclimatization period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 59 to 67%
- Air changes (per hr): air conditioned with adequate fresh air supply (12.9 to 14.0 air changes/hour).
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle.

IN-LIFE DATES: From: 30 March 2018 To: 12 July 2018

Route of administration:

oral: gavage

Vehicle:

other: Milli-Q water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 300 & 2000 mg/kg body weight (G1 and G2– First and second treatment steps)
- Amount of vehicle (if gavage):10 mL/kg

DOSAGE PREPARATION (if unusual): A required quantity (g) of test item was weighed in mortar and mixed using pestle by adding small volume of Milli-Q water until a uniform suspension was obtained. The mixture was quantitatively transferred to a measuring
cylinder. Further, a small volume of vehicle was added to the mortar and rinsed with the vehicle, 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 (mg/mL).

Doses:

G1 (FTS) - 300 mg/kg
G1 (STS) - 300 mg/kg
G2 (FTS) - 2000 mg/kg
G2 (STS) - 2000 mg/kg

No. of animals per sex per dose:

G1 (FTS) - 300 mg/kg - 3
G1 (STS) - 300 mg/kg - 3
G2 (FTS) - 2000 mg/kg - 3
G2 (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 - [300 mg/kg body weight - Treatment (FTS and STS)]:There were no pre-terminal deaths.
G2 - [2000 mg/kg body weight - Treatment (FTS and STS)]: There were no pre-terminal deaths.

Clinical signs:

other: G1 - [300 mg/kg body weight - Treatment (FTS and STS)]: There were no clinical sings observed in any of the rats. G2 - [2000 mg/kg body weight - Treatment (FTS and STS)]: There were no clinical signs observed in any of the rats.

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 Dose (mg/kg body weight)	Rat No.	Sex	Body weight (g)						Day of Death  (Time of Death)	No. dead/  No. tested	Pre-terminal deaths (%)
			Initial (Day 1)	8^thday	Weight change (day 8 – Initial)	15^thday	Weight change           (day 15 – Initial)	At Death
G1 (FTS) 300	Rm8749	F	155.28	202.69	47.41	215.76	60.48	NA	NA	0/3	0
	Rm8750	F	165.21	184.97	19.76	200.32	35.11	NA	NA
	Rm8751	F	142.08	166.44	24.36	178.43	36.35	NA	NA
G1 (STS) 300	Rm8752	F	162.46	180.12	17.66	172.30	9.84	NA	NA	0/3	0
	Rm8753	F	151.04	177.88	26.84	188.70	37.66	NA	NA
	Rm8754	F	143.05	154.52	11.47	172.67	29.62	NA	NA

F: Female        FTS: First Treatment Step            STS: Second Treatment Step                   NA: Not Applicable  0: No deaths 

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

  

Group and Dose (mg/kg body weight)	Rat No.	Sex	Body weight (g)						Day of Death  (Time of Death)	No. dead/  No. tested	Pre-terminal deaths (%)
			Initial (Day 1)	8^thday	Weight change (day 8 – Initial)	15^thday	Weight change           (day 15 – Initial)	At Death
G2 (FTS) 2000	Rm8755	F	150.86	159.21	8.35	178.35	27.49	NA	NA	0/3	0
	Rm8756	F	152.97	163.02	10.05	172.08	19.11	NA	NA
	Rm8757	F	151.21	161.78	10.57	176.14	24.93	NA	NA
G2 (STS) 2000	Rm8758	F	167.18	178.85	11.67	185.87	18.69	NA	NA	0/3	0
	Rm8759	F	156.58	164.03	7.45	178.69	22.11	NA	NA
	Rm8760	F	147.12	159.81	12.69	168.22	21.10	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 given test chemical in Wistar rats as per OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method).

The dose formulation was prepared by using Milli-Q water and administered as a single oral gavage to overnight fasted (16 to 18 hours) three female rats (G1-FTS) at the dose of 300 mg/kg body weight. There were no clinical signs of toxicity and pre-terminal deaths observed. As all the rats survived at this step, the test was confirmed with three additional female animals with the same dose of 300 mg/kg body weight (G1-STS). There were no clinical signs of toxicity and pre-terminal deaths observed at this step.

Based on the scheme - Annex 2c of the guideline OECD 423, the test was repeated at the dose of 2000 mg/kg body weight (G2-FTS). There were no clinical signs of toxicity observed. Hence, the test was confirmed with three additional female rats at the same dose of 2000 mg/kg body weight (G2-STS) as per the scheme - Annex 2c of the guideline OECD 423. There were no clinical signs of toxicity and pre-terminal deaths observed at this confirmatory test. Hence, the further dosing 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 again 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 acute oral 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: 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: Females: 213.42 to 248.13 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 rats were acclimatized for six, eight, twelve and fourteen days before treatment for dose range finding and main study respectively under standard laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 57 to 67 %
- Air changes (per hr): air conditioned with adequate fresh air supply (12.9 to 14.0 air changes/hour).
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle

IN-LIFE DATES: From: 30 March 2018 To: 29 June 2018

Type of coverage:

semiocclusive

Vehicle:

other: Milli-Q water

Details on dermal exposure:

TEST SITE
- Area of exposure: clipped skin of dorsolateral thoracic surface
- % coverage: 10% of the body surface
- Type of wrap if used: The applied area was covered with cotton gauze (size: Females: 8 x 5 cm of 6 ply) and it 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 formed a paste in Milli-Q water

Duration of exposure:

24 hours

Doses:

Two treatment group
G1 – DRF and Main

No. of animals per sex per dose:

DRF - 2000 mg/kg - 1
Main - 2000 mg/kg - 2

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 per the available literature, the LD50 of acute dermal median lethal dose of Rabbit is >5000 mg/kg body weight. Hence an intial dose of 2000 mg/kg body weight was tested with 1 female rat (dose range finding study). As there was no mortality at this dose the main study was conducted further with 2 animals to confirm the classification. There was no test item-related mortality. The subsequent dosing was done approximately 48 hours 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

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	Rm8761	F	231.51	244.34	12.83	250.66	19.15	0
G1 and 2000 Main study	Rm8762	F	248.73	248.81	0.08	256.20	7.47	0
	Rm8763	F	213.42	214.86	1.44	221.23	7.81	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) with the dose concentration of 2000 mg/kg in 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 application) 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 adhesive tape and cotton gauge 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:

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

The dose formulation was prepared by using Milli-Q water and administered as a single oral gavage to overnight fasted (16 to 18 hours) three female rats (G1-FTS) at the dose of 300 mg/kg body weight. There were no clinical signs of toxicity and pre-terminal deaths observed. As all the rats survived at this step, the test was confirmed with three additional female animals with the same dose of 300 mg/kg body weight (G1-STS). There were no clinical signs of toxicity and pre-terminal deaths observed at this step.

Based on the scheme - Annex 2c of the guideline OECD 423, the test was repeated at the dose of 2000 mg/kg body weight (G2-FTS). There were no clinical signs of toxicity observed. Hence, the test was confirmed with three additional female rats at the same dose of 2000 mg/kg body weight (G2-STS) as per the scheme - Annex 2c of the guideline OECD 423. There were no clinical signs of toxicity and pre-terminal deaths observed at this confirmatory test. Hence, the further dosing 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 again 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 acute oral 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".

Acute Inhalation toxicity: 

The acute toxicity inhalation study need not be conducted because exposure to humans via inhalation route is not likely taking into account the low vapour pressure of the substance, which is reported as 0.0000371 mm Hg at 25°C. Thus, exposure to inhalable dust, mist and vapour of the test 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) with the dose concentration of 2000 mg/kg in 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 application) 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 adhesive tape and cotton gauge 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 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 LD50 value is >2000 mg/kg bw, for acute oral toxicity 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 waiver was added so, not possible to classify.