N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxy-4-[[2-methoxy-5-methyl-4-[(methylamino)sulphonyl]phenyl]azo]naphthalene-2-carboxamide

Endpoint summary

Description of key information

RA from PB25

Under the conditions of the present study, a single oral application of the test item  to female rats at a dose of 2000 mg/kg body weight was associated with no sign of toxicity or mortality. The LD50 for female rats is  >  2000 mg/kg body weight

Under the conditions of the present study, single dermal application of the test item to rats at a dose of 2000 mg/kg body weight was associated with no mortality and neither signs of toxicity nor signs of irritation. The dermal LD50 was determined to be > 2000 mg/ kg body weight.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 06 MAR 2012 to 27 MAR 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 423) and according to GLP.

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

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: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 150-174 g
- Fasting period before study: 16-19 h
- Housing: in groups in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 110811)
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 0815), ad libitum
- Water: tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/-10
- Air changes (per hr): 10 x
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Details on oral exposure:

VEHICLE
- Amount of vehicle (if gavage): 10 mL / kg bw
- Justification for choice of vehicle: The vehivle was chosen due to its non-toxic characteristics
- Lot/batch no. (if required): MKBG0088V

MAXIMUM DOSE VOLUME APPLIED: 10 mL / kg bw

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: highest required dose tested.

Doses:

The starting dose was selected to be 2000 mg/kg bw. No compound-related mortality was recorded for any animal of step 1 or 2. Based on these results and according to the acute toxic class method regime no further testing was required.

No. of animals per sex per dose:

3 females per step / 2 steps performed

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were weighed on day 1 (prior to administratin) and on days 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight

Statistics:

no

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: no animal died during the 14-day observation period

Mortality:

All animals survived until the end of the study.

Clinical signs:

other: None of the animals showed any sign of toxicity.

Gross pathology:

With the exception of acute injection of blood vessels in the abdominal region, which is due to the euthanasia injection, no specific gross pathological changes were recorded for any animal.

Table: Body Weight Development - Absolute Body Weights in g and Body Weight Gain in %

Animal No. / Sex	g Day 1	g Day 8	g Day 15	% Day 1-15
Step 1 (2000 mg/kg Body Weight)
1 / female	166	192	193	16
2 / female	174	189	203	17
3 / female	150	167	170	13
Step 2 (2000 mg/kg Body Weight)
4 / female	156	179	184	18
5 / female	170	194	200	18
6 / female	166	183	194	17

Table: LD50 Cut-Off

Dose (unit)	Number of Animals Investigated	Number of Intercurrent Deaths	LD50 Cut-Off
2000 mg/kg bw	6	0	unclassified

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, a single oral application of the test item to female rats at a dose of 2000 mg/kg body weight was associated with no sign of toxicity or mortality. The LD50 for female rats is > 2000 mg/kg body weight

Executive summary:

Two groups, each of three female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight. The test item was suspended in cotton seed oil at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg.

All animals used in the study were allowed to acclimatise to the laboratory conditions for at least 5 days. The animals were observed on delivery, on inclusion in the study and before administration for mortality/morbidity and other clinical signs. All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. Their body weights were recorded on day 1 (prior to the administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

Table1:  Results per Step

Step	Sex/No.	Dose (mg/kg)	Number of Animals	Number of Intercurrent Deaths
1	female/1-3	2000	3	0
2	female/4-6	2000	3	0

All animals survived until the end of the study without showing any signs of toxicity.

Throughout the 14-day observation period, the body weight gain of the test animals was within the normal range of variation for this strain.

At necropsy, no macroscopic findings were observed in any animal of any step. The LD50 for rats is > 2000 mg/kg body weight

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

see Read-across justification in section 13 for details

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: no animal died during the 14-day observation period

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, a single oral application of the test item to female rats at a dose of 2000 mg/kg body weight was associated with no sign of toxicity or mortality. The LD50 for female rats is > 2000 mg/kg body weight

Executive summary:

Two groups, each of three female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight. The test item was suspended in cotton seed oil at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg.

All animals used in the study were allowed to acclimatise to the laboratory conditions for at least 5 days. The animals were observed on delivery, on inclusion in the study and before administration for mortality/morbidity and other clinical signs. All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. Their body weights were recorded on day 1 (prior to the administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

Table1:  Results per Step

Step	Sex/No.	Dose (mg/kg)	Number of Animals	Number of Intercurrent Deaths
1	female/1-3	2000	3	0
2	female/4-6	2000	3	0

All animals survived until the end of the study without showing any signs of toxicity.

Throughout the 14-day observation period, the body weight gain of the test animals was within the normal range of variation for this strain.

At necropsy, no macroscopic findings were observed in any animal of any step. The LD50 for rats is > 2000 mg/kg body weight

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 01 MAR 2012 to 04 MAY 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 402) and according to GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, 33178 Borchen, Germany
- Age at study initiation: males: 9 to 10 weeks; females 12 to 13 weeks
- Weight at study initiation: males: 229 to 241 g; females: 211-224 g
- Housing: The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 110811)
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 1114), ad libitum
- Water: tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals), ad libitum
- Acclimation period: Adequate acclimatisation period (at least five days) under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Air changes (per hr): 10 x / hour
- Photoperiod: 12 hours light, 12 hours dark
- Full barrier in an air-conditioned room
- Certificates of food, water and bedding are filed at BSL BIOSERVICE

Type of coverage:

semiocclusive

Vehicle:

other: cottonseed oil

Details on dermal exposure:

Preparation of the Animals:
The animals were marked for individual identification by tail painting.
Approximately 24 hours before the test, the fur was removed from the dorsal area of the trunk using an electric clipper.
Care was taken to avoid abrading the skin, and only animals with healthy intact skin were used.
No less than 10% of the body surface was cleared for the application.
Prior to the application a detailed clinical observation was made of all animals.

TEST SITE
The test item was applied at a single dose, uniformly over an area which was approximately 10% of the total body surface.
The test item was held in contact with the skin by a dressing throughout a 24-hour period.
- Type of wrap if used: The dressing consisted of a gauze-dressing and non-irritating tape and was fixed with an additional dressing in a suitable manner

REMOVAL OF TEST SUBSTANCE
- Washing (if done): residual test item was removed using the vehicle cottonseed oil
- Time after start of exposure: 24 hours (i.e. at the end of the exposure period)

VEHICLE
- Justification: The vehicle was chosen due to its minimal potential influence on irritation of the skin.
- Lot/batch no. (if required): MKBG0088V (Sigma Aldrich, expiry date: 2012-05)

Duration of exposure:

24 hours period

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

5

Control animals:

not required

Details on study design:

Observation period:
All animals were observed for 14 days after dosing

Weight Assessment:
The animals were weighed on day 1 (prior to the application) and on days 8 and 15.

Clinical Examination:
careful clinical examination was made several times on the day of dosing (at least once during the first 30 minutes
and with special attention given during the first 4 hours post-dose). As soon as symptoms were noticed they were recorded.
Thereafter, the animals were observed for clinical signs once daily until the end of the observation period. All abnormalities were recorded.
Cageside observations included changes in the skin and fur, eyes and mucous membranes. Also respiratory,
circulatory, autonomic and central nervous systems and somatomotor activity and behaviour pattern were examined.
Attention was directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Pathology:
At the end of the observation period the surviving animals were sacrificed with an overdosage of pentobarbital injected
intraperitoneally (Narcoren®, Merial) at the dosage of approximately 8 mL/kg bw. All animals were subjected to gross necropsy.
All gross pathological changes were recorded and in case of findings the tissues were preserved for a possible histopathological evaluation.
The preserved tissues of which no histopathological evaluation was made will be discarded 3 months after the release of the final report
unless otherwise agreed upon with the sponsor.

Evaluation of Results:
Individual reactions of each animal were recorded at each time of observation.
Toxic response data were recorded by sex and dose level.
Nature, severity and duration of clinical observations were described.
The body weight changes were summarised in a tabular form.
Necropsy findings were described.

Statistics:

According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results,
a statistical evaluation of the results is not regarded as necessary.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: no animal died during the 14-day observation period

Mortality:

No mortality was observed

Clinical signs:

other: No treatment-related effects were observed.

Gross pathology:

No treatment-related effects were observed.

Other findings:

No erythema or oedema was observed.

Table Absolute Body Weights in g and Body Weight Gain in %:

Dose: 2000 mg/kg body weight
Animal No. / Sex	g Day 1	g Day 8	g Day 15	% Day 1-15
21 / male	233	257	279	20
22 / male	231	251	281	22
23 / male	234	240	262	12
24 / male	241	261	285	18
25 / male	229	237	289	26
26 / female	211	212	221	5
27 / female	217	222	223	3
28 / female	221	219	225	2
29 / female	219	225	232	6
30 / female	224	223	228	2

Table LD50:

Dose (Unit)	Number of Animals Investigated	Number of Intercurrent Deaths	LD50
2000 mg/kg bw	5 males	0	> 2000 mg/kg bw
2000mg/kg bw	5 females	0	> 2000 mg/kg bw

bw = body weight

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, single dermal application of the test item to rats at a dose of 2000 mg/kg body weight was associated with no mortality and neither signs of toxicity nor signs of irritation. The dermal LD50 was determined to be > 2000 mg/ kg body weight.

Executive summary:

To test acute dermal toxicity 5 male and 5 female Wistar rats were subjected to a test according to OECD test guideline 402. The test material was applied once in cottonseed oil at a dose of 2000 mg/kg bw for 24 hours under semi-occlusive conditions to the skin of rats. During the 14 -day observation period none of the tested animals died, showed specific signs of systemic toxicity or revealed signs of dermal irritation. No findings were made at the macroscopic examination performed at the end of the observation period. The rather low weight gain (2 -6 % during the observation period) was not accounted for as adverse as there were no effects seen at the clinical and pathological examinations. Study authors mentioned that they might be secondary to the dressing. Under the conditions of the present study the dermal LD50 was determined to be > 2000 mg/kg body weight.

Reference 2

Endpoint:

acute toxicity: dermal

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

see Read-across justification in section 13 for details

Reason / purpose for cross-reference:

read-across source

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: no animal died during the 14-day observation period

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, single dermal application of the test item to rats at a dose of 2000 mg/kg body weight was associated with no mortality and neither signs of toxicity nor signs of irritation. The dermal LD50 was determined to be > 2000 mg/ kg body weight.

Executive summary:

To test acute dermal toxicity 5 male and 5 female Wistar rats were subjected to a test according to OECD test guideline 402. The test material was applied once in cottonseed oil at a dose of 2000 mg/kg bw for 24 hours under semi-occlusive conditions to the skin of rats. During the 14 -day observation period none of the tested animals died, showed specific signs of systemic toxicity or revealed signs of dermal irritation. No findings were made at the macroscopic examination performed at the end of the observation period. The rather low weight gain (2 -6 % during the observation period) was not accounted for as adverse as there were no effects seen at the clinical and pathological examinations. Study authors mentioned that they might be secondary to the dressing. Under the conditions of the present study the dermal LD50 was determined to be > 2000 mg/kg body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Justification for classification or non-classification

No classification, as no adverse effects were observed