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

Acute Toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

268-028-8

EC Name:

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

Cas Number:

67990-05-0

Molecular formula:

C32H25ClN4O5

IUPAC Name:

N-(5-chloro-2-methoxyphenyl)-3-hydroxy-4-[[2-methoxy-5-(phenylcarbamoyl)phenyl]diazenyl]-2-naphthamide

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.

Test animals

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

Administration / exposure

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.

Results and discussion

Effect levels

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.