Registration Dossier

Administrative data

Description of key information

For this enpoint, two key studies are available, one for oral and one for dermal toxicity. No data are available on acute toxicity via inhalation.

Acute oral toxicity:

The purpose of this study was to assess the oral acute toxicity of the test article when administered to rats (OECD 423, GLP). One group was treated with a single oral dose of 300 mg/kg bw. Two further groups were treated with an oral dose of 50 mg/kg bw (gavage). The median lethal dose of the test item after a single oral administration to female rats, observed over a period of 14 days is: LD50 cut-off (rat): 200 mg/ kg bw.

Acute dermal toxicity:

In order to determine the potential acute dermal toxicity of the test item, an acute dermal study on the rat was performed according to OECD 402 and in compliance to GLP. Wistar rats (5 male and 5 female) were exposed for 24h to 2000 mg/kg bw test substance.

The dermal LD50was 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
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-12-06 to 2016-12-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
2008
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
2002
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Wistar
Remarks:
WI(Han)
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
- 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 in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least five days) under laboratory conditions
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The test item was administered at a single dose by gavage using a feeding tube in the morning:
Step 1: 09:00 a.m.
Step 2: 08:15 a.m.
Step 3: 07:00 a.m.
The test item was administered at a dose volume of 10 mL/kg body weight.
Doses:
- step 1: 300 mg/kg body weight
- step 2: 50 mg/kg body weight
- step 3: 50 mg/kg body weight.
No. of animals per sex per dose:
- steps 1 to 3: 3 females each dose
Control animals:
no
Details on study design:
Preparation of the animals
The animals were marked for individual identification by tail painting. Prior to the administration a detailed clinical observation was made of all animals. Only healthy animals were used. 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.

Observation Period
The surviving 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
Animals sacrificed for ethical reasons during the observation period were necropsied as soon as they were killed.
At the end of the observation period the surviving animals were sacrificed with an overdosage of pentobarbital injected intraperitoneally at a dosage of 250-400 mg/kg bw. All animals were subjected to gross necropsy. All gross pathological changes were recorded but not preserved for possible histopathological evaluation.

Evaluation of Results
Results were interpreted according to OECD Guideline 423, Annex 2 and GHS. Individual reactions of each animal were recorded at each time of observation. Toxic response data were recorded by 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:
n.a.
Key result
Sex:
female
Dose descriptor:
LD50 cut-off
Effect level:
200 mg/kg bw
Based on:
test mat.
Mortality:
- At a concentration of 300 mg/kg all animals had to be sacrificed within the first hours after dose administration.
- At a concentration of 50 mg/kg one of six animals died within the first five hours after dose administration.
Clinical signs:
The test item showed mortality and other acute oral toxicity characteristics after a single dose administration. Animals exhibited moderately reduced spontaneous activity, opisthotonos, severe ataxia, moderate piloerection, cyanosis and half eyelid closure within the first four hours after application of the test item.
Body weight:
None of the animals showed weight loss during the observation period
Gross pathology:
All animals treated with 300 mg/kg bw showed a bloody stomach, duodenum, jejunum, ileum and peyer’s patches, one animal treated with 50 mg/kg bw showed bloody stomach, duodenum, jejunum, ileum and peyer’s patches.

LD50 Cut-Off

Starting Dose (mg/kg bw) Number of animals Number of intercurrent deaths LD50 Cut-Off (mg/kg bw)
300 3 3 200
50 6 1

bw = body weight

Interpretation of results:
Category 3 based on GHS criteria
Conclusions:
The median lethal dose of the test item after a single oral administration to female rats, observed over a period of 14 days is: LD50 cut-off (rat): 200 mg/kg bw.
Executive summary:

The purpose of this study was to assess the toxicity of the test article when administered to rats after a single oral dose. The study was carried out in accordance with OECD TG 423 and in compliance to GLP.

One group, each of three female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 300 mg/kg body weight. The test item was dissolved in aqua ad injectionem (sterile water) at a concentration of 0.03 g/mL and administered at a dose volume of 10 mL/kg. Two further groups, each of three female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 50 mg/kg body weight. The test item was suspended with the vehicle aqua ad injectionem (sterile water) at a concentration of 0.005 g/mL and administered at a dose volume of 10 mL/kg.

All animals used in the study after their entrance at the test facility 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.

All animals treated with the test item at a dose of 300 mg/kg had to be sacrificed for ethical reasons on test day 1. One animal treated with the test item at a dose of 50 mg/kg died spontaneously on the day of treatment. All other animals treated with the test item at a dose of 50 mg/kg survived until the end of the study showing test-item related signs of toxicity.

The most relevant clinical findings in the animals treated with the test item at a dose of 300 mg/kg bw were reduced spontaneous activity, prone position, ataxia, slow movements, piloerection, recumbency, opisthotonos, cyanosis and half eyelid closure.

The most relevant clinical findings in the animals treated with the test item at a dose of 50 mg/kg bw were reduced spontaneous activity, hunched posture, prone position, ataxia, piloerection, abnormal breathing, eyes closed and half eyelid closure. All symptoms recovered within up to 1 day post-dose.

Macroscopic findings of animals not having survived until the end of the observation period: Necropsy revealed a bloody stomach and a bloody intestine in all animals (No. 1-3, 9). Macroscopic findings of surviving animals: At necropsy, no treatment-related macroscopic findings were observed in any animal (no. 4-6, 7+8).

Under the conditions of the present study, a single oral application of the test item to rats at a dose of 300 mg/kg body weight was associated with signs of toxicity or mortality. The median lethal dose of the test item after a single oral administration to female rats, observed

over a period of 14 days is: LD50 cut-off (rat): 200 mg/ kg bw.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
200 mg/kg bw
Quality of whole database:
The study was carried out in accordance with OECD TG 423 and in compliance to GLP.

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:
experimental study
Adequacy of study:
key study
Study period:
2017-06-12 to 2017-07-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The test for acute dermal toxicity is performed on the rat. Although several mammalian species may be used, the rat is the preferred rodent species.
In the assessment and evaluation of the toxic characteristics of chemicals, the determination of acute dermal toxicity is useful where exposure by the dermal route is likely.
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
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test animals:
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female (non-pregnant and nulliparous)
Number of animals: 5 male and 5 female animals
Age at the beginning of the study: males: 11-12 weeks; females: 12-13 weeks
Body weight on the day of administration:Males: 276g - 304g; Females: 218g - 230g

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
- 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
- Adequate acclimatisation period (at least five days) under laboratory conditions
Type of coverage:
occlusive
Vehicle:
water
Details on dermal exposure:
Preparation of the Animals:
- The animals were marked individualy by tail painting
- Approx. 24 hours before the test, the fur was removed from the dorsal area of the trunk using an electric clipper. Only animals with healthy intact skin were used
- Prior to the application a detailed clinical observation was performed on all animals. Only healthy animals were used.

Application:
- 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

Dose Level:
- The test item was applied at a single dose of 2000 mg/kg body weight to each animal

Exposure Period:
- 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 aqua ad injectionem



Duration of exposure:
24 h
Doses:
a single dose of 2000 mg/kg bw
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Details on study design:
Observation Period:
- All animals were observed for 14 days after dosing

Evaluation of Primary Skin Irritation:
- Signs of erythema and oedema were assessed using the scoring system laid down in OECD Guideline 404

Weight Assessment:
The animals were weighed on day 1 (prior to the application) 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. Attention was directed to observations of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma

Pathology:
- At the end of the observation period the animals were sacrificed with an overdosage of pentobarbital injected intraperitoneally at a dosage of 250-400 mg/kg bw
- All animals were subjected to gross necropsy and examined macroscopically for gross pathological changes
- In absence of gross pathological changes no tissues were preserved for a possible histopathological evaluation

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:
not specified
Preliminary study:
not applicable
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: skin irritation reported
Sex:
male
Dose descriptor:
other: dose
Effect level:
2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no mortality
Sex:
female
Dose descriptor:
other: dose
Effect level:
2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: no mortality
Mortality:
The test item showed no mortality
Clinical signs:
No signs of systemic toxicity. Signs of local toxicity and irritation.
Body weight:
- A slight weight loss was recorded for 3 out of 10 animals during the two weeks of observation
- 1 out of 5 males showed unchanged weight during the first week
-1 out of 5 females showed unchanged weight during the first week
-1 out of 5 females showed unchanged weight during first and second week
- All other animals showed weight gain during first and second week

The effects on weight development might be secondary to the dressing, and toxicological relevance of this finding cannot clearly be concluded.
Gross pathology:
No specific gross pathological changes were recorded for any animal
Other findings:
- Erythema grade 1 was observed in all male animals but no female animals
- Crust was observed in 4 of 5 male and all female animals
- Necrosis was observed in 3 of 5 male animals but in no female animals
- Necrosis was not reversible within the observation period

Absolute body weights in g and body weight gain in %

Animal No. / Sex body weight gain (g) %
Day 1 Day 8 Day 15 Day 1-15
21/Male 286 271 307 7
22/Male 304 315 349 15
23/Male 279 295 327 17
24/Male 276 283 304 10
25/Male 287 291 316 10
26/Female 219 216 215 -2
27/Female 218 224 230 6
28/Female 229 227 245 7
29/Female 230 240 240 4
30/Female 223 224 230 3

The effects on weight loss are a common observation for animals within this study type, mainly for female animals and might be secondary to the dressing, and toxicological relevance of this finding cannot clearly be concluded.

 LD50Cut-Off

Dose (mg/kg bw)

Number of Animals

Number of Intercurrent Deaths

LD50Cut-Off (mg/kg bw)

2000

5 males

0

> 2000

2000

5 females

0

> 2000

bw = body weight

Interpretation of results:
GHS criteria not met
Conclusions:
The dermal lethal dose of the test item after a single dermal administration to female rats, observed over a period of 14 days is: LD50 cut-off (rat) > 2000 mg/kg bw.
Executive summary:

In order to determine the potential acute dermal toxicity of the test item, an acute dermal study on the rat was performed according to OECD 402 and in compliance to GLP. Wistar rats (5 male and 5 female) were exposed for 24h to 2000 mg/kg bw test substance.

Single dermal application of the test item to rats at a dose of 2000 mg/kg body weight was not associated with mortality but with signs of local toxicity and irritation were observed. Although weight loss effects were observed, especially with female animals, it is concluded that this is a common observation for this study type and that this is probably secondary to the dressing. Toxicological relevance of this finding cannot be clearly concluded.

In conclusion, the dermal LD50was 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
Quality of whole database:
no issues with the quality of the data

Additional information

Acute oral toxicity:

The purpose of this study was to assess the toxicity of the test article when administered to rats after a single oral dose (in accordance with OECD 423, GLP).

One group, each of three female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 300 mg/kg bw. Two further groups, each of three female WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 50 mg/kg bw. All animals treated with the test item at a dose of 300 mg/kg bw had to be sacrificed for ethical reasons on test day 1. One animal treated with the test item at a dose of 50 mg/kg bw died spontaneously on the day of treatment. All other animals treated with the test item at a dose of 50 mg/kg bw survived until the end of the study showing test-item related signs of toxicity.

The most relevant clinical findings in the animals treated with the test item at a dose of 300 mg/kg bw were reduced spontaneous activity, prone position, ataxia, slow movements, piloerection, recumbency, opisthotonos, cyanosis and half eyelid closure. The most relevant clinical findings in the animals treated with the test item at a dose of 50 mg/kg bw were reduced spontaneous activity, hunched posture, prone position, ataxia, piloerection, abnormal breathing, eyes closed and half eyelid closure. All symptoms recovered within up to 1 day post-dose.

Macroscopic findings of animals not having survived until the end of the observation period: Necropsy revealed a bloody stomach and a bloody intestine in all animals. Macroscopic findings of surviving animals: At necropsy, no treatment-related macroscopic findings were observed in any animal.

Under the conditions of the present study, a single oral application of the test item to rats at a dose of 300 mg/kg body weight was associated with signs of toxicity or mortality. The median lethal dose of the test item after a single oral administration to female rats, observed over a period of 14 days is: LD50 cut-off (rat): 200 mg/ kg bw.

Acute dermal toxicity:

In order to determine the potential acute dermal toxicity of the test item, an acute dermal study on the rat was performed according to OECD 402 and in compliance to GLP. Wistar rats (5 male and 5 female) were exposed for 24h to 2000 mg/kg bw test substance.

Single dermal application of the test item to rats at a dose of 2000 mg/kg bw was not associated with mortality but with signs of local toxicity and irritation was observed. Although weight loss effects were observed, especially with female animals, it is concluded that this is a common observation for this study type and that this is probably secondary to the dressing. Toxicological relevance of this finding cannot be clearly concluded.

In conclusion, the dermal LD50was determined to be > 2000 mg/kg bw.

Justification for classification or non-classification

Acute oral toxicity

The median lethal dose of Ingrain Blue 2.2 after a single oral administration to female rats, observed over a period of 14 days is:

LD50 cut-off (rat): 200 mg/ kg body weight. According to Annex I of Regulation (EC) 1272/2008 the test item is classified into "Category 3".

Acute dermal toxicity

The potential acute dermal toxicity of the test item on the rat was tested. 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 but with signs of local toxicity and irritation. The dermal LD50 was determined to be > 2000 mg/ kg body weight. Hence, the substance should not be classified.