N-(5-chloro-2-methoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-[(phenylamino)carbonyl]phenyl]azo]naphthalene-2-carboxamide

Endpoint summary

Description of key information

Oral (OECD 423): LD50 (rat) > 2000 mg/kg bw

Dermal (OECD 402): LD50 (rat) > 2000 mg/kg bw (read-across from analogue substance Pigment Red 170 [4-[[4-(aminocarbonyl)phenyl]azo]-N-(2-ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide])

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

Study period:

From 8 MAR 2012 to 17 MAY 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 423), GLP compliant

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

Specific details on test material used for the study:

In October 2011 the EU commission published a recommendation (2011/696/EU) on the definition of nanomaterials. From the results of analyses it is concluded that the registered substance C.I.Pigment Red 269 falls within the boundaries of the nanomaterial definition. Hence, studies were performed using the nanomaterial.

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0815 for steps 1 and 2, 0715 for step 3)
- Free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control,
microbiological controls at regular intervals)
- The animals were kept in groups / individually in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
(lot no. 110811 for steps 1 and 2, 261111 for step 3)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions

Route of administration:

oral: gavage

Details on oral exposure:

The animals were marked for individual identification by tail painting.
Prior to the administration a detailed clinical observation was made of all animals.
Prior to the administration food was withheld from the test animals for 16 to 19 hours (access to water was permitted).
Following the period of fasting the animals were weighed and the test item was administered. Food was provided again
approximately 4 hours post dosing.
The test item was administered at a single dose by gavage using a feeding tube.
The test item was administered at a dose volume of 10 mL/kg body weight.

Doses:

Since no or only a very low acute toxicity was expected the starting dose was selected to be 2000 mg/kg body weight.
No compound-related mortality was recorded for any animal of step 1 or 2. However, no correction to the active ingredient
content (i.e. content C.I.) was made in these two steps. In order to assess the toxicity of the active ingredient at a dose of 2000 mg/kg
as initially planned, three additional animals were treated at 2000 mg/kg with correction to the active ingredient content (i.e. content C.I.).
No compound-related mortality and no adverse clinical signs were recorded for any animal of step 3. Based on these results and for
animal welfare reason the second confirming step at 2000 mg/kg body weight with correction to the active ingredient content (i.e. content C.I.)
of the test item was not performed. The data collected were sufficient to assess the potential toxicity of the test item.

No. of animals per sex per dose:

3

Control animals:

no

Details on study design:

Observation Period
All animals were observed for 14 days after dosing for general clinical signs, morbidity and mortality.

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

Clinical Examination
A 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.
Particular attention was directed to observations of tremor, 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; lot no.: 218121; expiry date: 12/2014) at a 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.

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:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

other: content C.I.

Remarks on result:

other: no animal died within the 14 day observation period

Mortality:

All animals survived until the end of the study.

Clinical signs:

other: No signs of toxicity were observed in any animal.

Gross pathology:

At necropsy, no macroscopic findings were observed in any animal of any step.

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 (1860 mg/kg Body Weight)
1 / female	154	182	199	29
2 / female	161	187	197	22
3 / female	145	172	188	30
Step 2 (1860 mg/kg Body Weight)
4 / female	156	180	190	22
5 / female	163	195	201	23
6 / female	161	190	196	22
Step 3 (2000 mg/kg Body Weight)
7 / female	184	207	212	15
8 / female	188	205	228	21
9 / female	176	189	205	16

Table: LD50 Cut-Off

Dose (unit)	Number of Animals Investigated	Number of Intercurrent Deaths	LD50 Cut-Off
1860 mg/kg bw	6	0	unclassified
2000 mg/kg bw	3	0	unclassified

bw = body weight

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the present study, a single oral application of the test item to rats at a dose of 2000 mg/kg body weight was associated with no signs of toxicity or mortality. The median lethal dose of the active ingredient after a single oral administration to female rats, observed over a period of 14 days is greater than 2000 mg/kg body weight.

Executive summary:

Acute oral toxicity was investigated according to OECD guideline 423. 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 1860 mg/kg body weight (no correction to the active ingredient content (i.e. content C.I.) was made). The test item was suspended in the vehicle cottonseed oil at a concentration of 0.186 g/mL and administered at a dose volume of 10 mL/kg.

One group of three female WISTAR Crl: WI(Han) rats, was treated with the test item by oral gavage administration at a dosage

of 2000 mg/kg body weight considering the active ingredient content (i.e. content C.I.) of the test item. The test item was suspended in the vehicle cottonseed 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 after their entrance at BSL 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.

Results per Step

Step	Sex/No.	Dose (mg/kg)	Number of Animals	Number of Intercurrent Deaths
1	female/1-3	1860	3	0
2	female/4-6	1860	3	0
3	female/7-9	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 oral LD50 in rats is > 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 1) study performed with the registered substance. The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VII, Item 8.5, of Regulation (EC) No. 1907/2006 (REACH).

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

From 02 MAR 2012 to 08 MAY 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD 402), GLP-compliant

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:

- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3 °C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1114)
- Free access to tap water, sulphur acidified to a pH value of approximately 2.8
(drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept individually in IVC cages, type III H, polysulphone cages on Altromin
saw fibre bedding (lot no. 110811)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least five days) under laboratory conditions

Type of coverage:

semiocclusive

Vehicle:

cotton seed 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.

Application:
The test item was applied at a single dose, uniformly over an area which was approximately 10% of the total body surface. In order to ensure good skin contact the test item was moistened with the vehicle.
The test item was held in contact with the skin by a dressing throughout a 24-hour period.
The dressing consisted of a gauze-dressing and non-irritating tape and was fixed with an additional dressing in a suitable manner.

Duration of exposure:

The test item was held in contact with the skin throughout a 24-hour period. At the end of the exposure period the residual test item was removed using cottonseed oil.

Doses:

The test item was applied at a single dose of 2000 mg/kg body weight to each animal.

No. of animals per sex per dose:

5 male and 5 female

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:

other: Content C.I.

Remarks on result:

other: no animal died within 14 d 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. Scratches were observed in 1 of 5 male animals.

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	227	242	276	22
22 / male	236	251	277	17
23 / male	229	248	278	21
24 / male	230	241	274	19
25 / male	245	260	280	14
26 / female	216	220	223	3
27 / female	213	214	221	4
28 / female	218	215	217	-0.50
29 / female	215	218	223	4
30 / female	207	211	220	6

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:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

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 neither mortality nor signs of toxicity. In one male animal scratches were observed from day 6 until the end of the observation period which might be caused by scratching activities of the animal. The dermal LD50 was determined to be > 2000 mg / kg body weight.

Executive summary:

Acute toxicity after dermal application was investigated in Wistar rats according to an OECD 402 limit test. Animals (5 males and 5 females) were applied 2000 mg/kg bw in cottonseed oil for 24 hours. No animal died during the observation period.

Table1:  Results per Step

Sex	Dose (mg/kg bw)	Number of Animals	Number of Intercurrent Deaths
male	2000	5	0
female	2000	5	0

Signs of toxicity related to dose level used, time of onset and duration:

No treatment-related effects were observed

Effect on organs (related to dose level):

No treatment-related effects were observed.

Signs of irritation:

No erythema or oedema was observed. Scratches were observed in 1 of 5 male animals.

Conclusion

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 neither mortality nor signs of toxicity. In one male animal scratches were observed from day 6 until the end of the observation period which might be caused by scratching activities of the animal.

The dermal LD50 was determined to be > 2000 mg / kg body weight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The available information comprises an adequate and reliable (Klimisch score 1) study from an analogue substance with similar structure and intrinsic properties. Read-across is justified based on a common metabolic breakdown of target and source substances and consistent trends in environmental fate, ecotoxicological and toxicological properties. The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII, Item 8.5, in accordance with Annex XI, Item 1.5, of Regulation (EC) No. 1907/2006.

Additional information

Acute oral toxicity

Acute oral toxicity was investigated in a study performed according to OECD guideline 423 with the registered (target) substance Pigment Red 269 (BSL_120849A). One group of three female WISTAR Crl: WI(Han) rats, was treated with the test item by oral gavage administration at a dosage of 2000 mg/kg bw considering the active ingredient content (i.e. content C.I.) of the test item. The test item was suspended in the vehicle cottonseed oil at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg bw. All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. 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 oral LD50 value in rats is > 2000 mg/kg bw.

Acute dermal toxicity

Acute toxicity after dermal application was investigated in Wistar rats according to an OECD guideline 402 limit test with the analogue substance Pigment Red 170 (4-[[4-(aminocarbonyl)phenyl]azo]-N-(2-ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide) (BSL_120508). A dose of 2000 mg/kg bw in cottonseed oil was applied to the clipped skin of 5 males and 5 females under semiocclusive conditions for 24 h. No animal died during the observation period. No erythema or oedema was observed. Scratches were observed in 1/5 male animals from Day 6 until the end of the observation period which might be caused by scratching activities of the animal. The dermal LD50 value was determined to be > 2000 mg/kg bw.

Justification for classification or non-classification

The available data on acute oral toxicity obtained with the registered substance and on acute dermal toxicity derived from an analogue substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.