7-amino-3-{(E)-[5-({4-(2-chloroethyl)butanoyl}amino)]diazenyl}-4-hydroxy-8-[(E)-(4-{2-(sulfonatooxy)ethyl}) diazenyl]naphthalene, polysulfonate, polysulfonyl, polyphenyl, sodium/potassium salt

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 January 2012 to 09 February 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

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 402 (Acute Dermal Toxicity)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Test type:

standard acute method

Limit test:

yes

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species/strain: healthy rats, WISTAR rats Crl: WI(Han) (full barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; The female animals were non-pregnant and nulliparous.
Number of animals: 5 male and 5 female
Age at the beginning of the study: males: 8 – 9 weeks, females: 11 – 12 weeks
Body weight on the day of administration: males: 232 – 254 g; females: 216 – 219 g
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the
German Act on Animal Welfare the animals were bred for experimental purposes.

ENVIROMENTAL CONDITION

- 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)
- 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

Administration / exposure

Type of coverage:

semiocclusive

Vehicle:

water

Remarks:

Diprom, lot no. 10952-1, expiry date: 09/2013

Details on dermal exposure:

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. 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 tap water.

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.
On the basis of the test results, the test item may be classified in one of the following classes in conformity with the criteria given in Annex VI to Commission Directive 2001/59/EC:
• Very toxic
Substances and preparations shall be classified as very toxic, and assigned the symbol “T+” and indication of danger “very toxic” in accordance with the criteria specified below:
R27 Very toxic in contact with skin
- LD50 dermal, rat or rabbit: <= 50 mg/kg
• Toxic
Substances and preparations shall be classified as toxic, and assigned the symbol “T” and indication of danger “toxic” in accordance with the criteria specified below. Risk phrases shall be assigned in accordance with the following criteria:
R24 Toxic in contact with skin
- LD50 dermal, rat or rabbit: 50 < LD50 <= 400 mg/kg
• Harmful
Substances and preparations shall be classified as harmful, and assigned the symbol “Xn” and indication of danger “harmful” in accordance with the criteria specified below. Risk phrases shall be assigned in accordance with the following criteria:
R21 Harmful in contact with skin
- LD50 dermal, rat or rabbit: 400 < LD50 <= 2000 mg/kg
On the basis of the test results, the following risk phrases may be assigned in conformity with the criteria given in Annex I of Regulation (EC) 1272/2008:
Category 1: LD50 <= 50 mg/kg. DANGER. Skull and crossbones in diamond. Fatal in contact with skin.
Category 2: LD50 > 50 mg/kg <= 200 mg/kg. DANGER. Skull and crossbones in diamond. Fatal in contact with skin.
Category 3: LD50 > 200 mg/kg <= 1000 mg/kg. DANGER. Skull and crossbones in diamond. Toxic in contact with skin.
Category 4: LD50 > 1000 mg/kg <= 2000 mg/kg. WARNING. Exclamation point in diamond. Harmful in contact with skin.
On the basis of the test results, the following risk phrases may be assigned in conformity with the criteria given in GHS - Globally Harmonized System of Classification and Labelling of Chemicals, third revised edition, July 2009:
Category 1: LD50 <= 50 mg/kg. DANGER. Skull and crossbones in diamond. Fatal in contact with skin.
Category 2: LD50 > 50 mg/kg <= 200 mg/kg. DANGER. Skull and crossbones in diamond. Fatal in contact with skin.
Category 3: LD50 > 200 mg/kg <= 1000 mg/kg. DANGER. Skull and crossbones in diamond. Toxic in contact with skin.
Category 4: LD50 > 1000 mg/kg <= 2000 mg/kg. WARNING. Exclamation point in diamond. Harmful in contact with skin.
Category 5: LD50 > 2000 mg/kg <= 5000 mg/kg. WARNING. No symbol. May be harmful in contact with skin.

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:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality was observed.

Clinical signs:

other: The most relevant clinical findings in the animals treated with the test item at a dose of 2000 mg/kg bw were reduced spontaneous activity, piloerection and nasal discharge. These signs are considered to be common symptoms in acute dermal tests.

Gross pathology:

No treatment-related effects were observed.

Other findings:

No erythema or oedema was observed. Eschar was observed in 1 of 5 male and 1 of 5 female animals.
All signs of irritation were reversible within the observation period.

Any other information on results incl. tables

Table Clinical Signs of Systemic Toxicity – Individual Data - Males:

Animal No. / Sex / Dose	Time of Observation (Post-Dose)	Observations (for Signs of Dermal Irritation, see Table 5)
21/ male / 2000 mg/kg bw	0 min, 30 min	no signs of toxicity
	1 h	nasal discharge
	2 h until the end of the observation period	no signs of toxicity
22/ male / 2000 mg/kg bw	0 min, 30 min	no signs of toxicity
	1 h	nasal discharge
	2 h until the end of the observation period	no signs of toxicity
23/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
24/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
25/ male / 2000 mg/kg bw	during the whole observation period	no signs of toxicity

min = minute(s), h = hour(s), bw = body weight; day1 = day of administration

Table Clinical Signs of Systemic Toxicity – Individual Data – Females:

Animal No. / Sex / Dose	Time of Observation (Post-Dose)	Observations (for Signs of Dermal Irritation, see Table 6)
26/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity
27/ female / 2000 mg/kg bw	day 1	no signs of toxicity
	day 2	slightly reduced spontaneous activity, slight piloerection
	day 3 until the end of the observation period	no signs of toxicity
28/ female / 2000 mg/kg bw	day 1	no signs of toxicity
	day 2	slightly reduced spontaneous activity
	day 3 until the end of the observation period	no signs of toxicity
29/ female / 2000 mg/kg bw	0 min, 30 min	no signs of toxicity
	1 h	nasal discharge
	2 h until the end of the observation period	no signs of toxicity
30/ female / 2000 mg/kg bw	during the whole observation period	no signs of toxicity

min = minute(s), h = hour(s), bw = body weight;day1 = day of administration

Table Skin Irritation – Individual Data – Males:

Day after Start of Application	Animal No. 21		Animal No. 22		Animal No. 23		Animal No. 24		Animal No. 25
	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments
day 2	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 3	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 4	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 5	0/0	*	0/0	es,*	0/0	*	0/0	*	0/0	*
day 6	0/0	*	0/0	es,*	0/0	*	0/0	*	0/0	*
day 7	0/0	*	0/0	es,*	0/0	*	0/0	*	0/0	*
day 8	0/0	*	0/0	es,*	0/0	*	0/0	*	0/0	*
day 9	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 10	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 11	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 12	0/0	nsf	0/0	*	0/0	*	0/0	*	0/0	nsf
day 13	0/0	nsf	0/0	*	0/0	*	0/0	*	0/0	nsf
day 14	0/0	nsf	0/0	*	0/0	*	0/0	*	0/0	nsf
day 15	0/0	nsf	0/0	*	0/0	*	0/0	nsf	0/0	nsf
Comments: E = erythema; O = oedema; 1, 2, 3, 4 = scoring system laid down in OECD Guideline 404 (Table 2) es = eschar; nsf = no specific findings; * = red coloured residue of the test item

Table Skin Irritation – Individual Data – Females:

Day after Start of Application	Animal No. 26		Animal No. 27		Animal No. 28		Animal No. 29		Animal No. 30
	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments
day 2	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 3	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 4	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 5	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 6	0/0	*	0/0	*	0/0	es, *	0/0	*	0/0	*
day 7	0/0	*	0/0	*	0/0	es, *	0/0	*	0/0	*
day 8	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 9	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 10	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 11	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 12	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 13	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 14	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 15	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
Comments: E = erythema; O = oedema; 1, 2, 3, 4 = scoring system laid down in OECD Guideline 404 (Table 2) es = eschar; nsf = no specific findings; * = red coloured residue of the test item

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	238	246	276	16
22 / male	244	249	277	14
23 / male	254	266	298	17
24 / male	248	251	290	17
25 / male	232	240	265	14
26 / female	216	213	220	2
27 / female	216	214	226	5
28 / female	217	209	218	0
29 / female	219	209	214	-2
30 / female	219	207	214	-2

Table Macroscopic Findings - Individual Data – Males and Females:

Dose: 2000 mg/kg bw
Animal No. / Sex	Organ	Macroscopic Findings
21 / male	-	nsf
22 / male	-	nsf
23 / male	-	nsf
24 / male	-	nsf
25 / male	-	nsf
26 / female	-	nsf
27 / female	-	nsf
28 / female	-	nsf
29 / female	-	nsf
30 / female	-	nsf

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

Applicant's summary and conclusion

Interpretation of results:

Category 5 based on GHS criteria

Remarks:

Migrated information

Conclusions:

Under the conditions of the present study, single dermal application of the test item FAT 40858/A TE to rats at a dose
of 2000 mg/kg body weight was associated with no mortality but signs of toxicity (which are considered to be common symptoms
in acute dermal tests) and signs of irritation. The dermal LD50 was determined to be > 2000 mg FAT 40858/A TE / kg body weight.
In conformity with the criteria given in Annex VI to Commission Directive 2001/59/EC the test item FAT 40858/A TE has no obligatory
labelling requirement for percutaneous toxicity.
According to Annex I of Regulation (EC) 1272/2008 the test item FAT 40858/A TE has no obligatory labelling requirement for
percutaneous toxicity and is unclassified.
According to GHS (Globally Harmonized Classification System) the test item FAT 40858/A TE has no obligatory labelling requirement
for percutaneous toxicity and is classified into Category 5.

Executive summary:

On the basis of the test results given below and in conformity with the criteria given in Annex VI to Commission Directive 2001/59/EC, the substance should be:

classified asvery toxic
classified astoxic
classified as harmful
not classified	X
limit test	X

On the basis of the test results given below and in conformity with the criteria given inAnnex I of Regulation (EC) 1272/2008, the substance should be:

classified into category 1
classified into category 2
classifiedinto category 3
classifiedinto category 4
not classified	X

On the basis of the test results given below and in conformity with the criteria given inGHS (Globally Harmonized System of Classification and Labelling of Chemicals), the substance should be:

classified into category 1
classified into category 2
classifiedinto category 3
classifiedinto category 4
classifiedinto category 5	X
not classified

LD₅₀:  > 2000 mg /kg bw

Species/strain:   WISTAR Crl: WI(Han) rats

Vehicle (moistening): aqua ad injectionem

Number of animals:  5 male and 5 female

Duration of exposure: 24 hours

Method:  OECD 402, EC 440/2008, Method B.3, OPPTS 870.1200

 

Table:  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:

The most relevant clinical findings in the animals treated with the test item at a dose of 2000 mg/kg bw were reduced spontaneous activity, piloerection and nasal discharge. These signs are considered to be common symptoms in acute dermal tests.

Effect on organs (related to dose level): No treatment-related effects were observed.

Signs of irritation: No erythema or oedema was observed. Eschar was observed in 1 of 5 male and 1 of 5 female animals.

All signs of irritation were reversible within the observation period.