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Description of key information

In the acute oral toxicity study in rats, signs of unspecific toxicity were observed at dose levels of 100 to 800 mg/kg bw. The oral LD50 for males and females together was calculated to be 216 mg/kg bw (CI 172 to 272 mg/kg bw) based on test material. This calculated to 124 mg/kg bw for the active ingredient.

No toxicity was obderved in the acute dermal toxicity study, leading to a dermal LD50 of above 2000 mg/kg bw.

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:
02 Nov -13 Dec 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: 9 wks (male), 14 wks (female)
- Weight at study initiation: mean 181 g (male), 176 g (female)
- Fasting period before study: 16 h
- Housing: Makrolon Type III cages (group of 5 animals)
- Diet (e.g. ad libitum): ad lib.
- Water (e.g. ad libitum): ad lib.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 60 rh
- Light/darkness (h): 12/12
- Acclimation period: min. 5 days
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Application volume: 10 mL/kg bw
Doses:
males: 50, 100, 200, 250, 500, 800 mg/kg bw
females: 50, 100, 200, 250, 310 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: daily
- Other examinations performed: clinical signs, body weight, gross pathology
Statistics:
Probit analysis
Preliminary study:
Probit analysis
Sex:
male/female
Dose descriptor:
LD50
Effect level:
216 mg/kg bw
Based on:
test mat.
95% CL:
172 - 272
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
124 mg/kg bw
Based on:
act. ingr.
95% CL:
99 - 156
Mortality:
50 mg/kg bw: no mortality
100 mg/kg bw: 0-20%
200 mg/kg bw: 20%
250 mg/kg bw: 80%
310 (female) & 800 (male) mg/kg bw: 100%
Mortality occurred between 1h - 1d after application
Clinical signs:
50 mg/kg bw: no symptoms
100 - 800 mg/kg bw: bad general condition, sedation, prone or later position, bristled fur
200 -800 mg/kg bw: algospams
310 / 800 mg/kg bw: gasping
All symptoms occurred ca. 30 min after application and disappeared until day 2.
Body weight:
Males: no influence
Females: slight growth inhibition from 200 mg/kg bw
Gross pathology:
100 - 800 mg/kg bw: stomach mucosa elapsed, stomach/gut contents reddened and bloated
250 - 800 mg/kg bw: lungs reddened
All survivors from 50 - 500 mg/kg bw were pathologically w/o finding
Interpretation of results:
Category 3 based on GHS criteria
Conclusions:
The LD50 (oral, gavage) in male and female rats was determined to be 124 mg/kg bw. The substance is classified as Category 3.
Executive summary:

An acute oral toxicity study was carried out with the test item in male and female rats. Five rats/sex/dose groups received a single gavage of the test item dissolved in distilled water at dose levels of 50, 100, 200, 250, 500, 800 mg/kg bw for males and 50, 100, 200, 250, 310 mg/kg bw for females. The animals were observed for signs of toxicity for 14 days thereafter.

Clinical signs observed in animals after a single administration of 100 to 800 mg/kg bw Basic Blue 159 were poor general condition, piloerection, sedation and prone or lateral position. Occasionally, tonic-clonic convulsions, laboured respiration and stilted gait occurred. A slight delay in body weight development was observed in females at a dose of 200 mg/kg bw in the first week of the study. In the pathological-anatomical examination of the deceased male and female rats at doses of 100 to 800 mg/kg bw, non-specific damage to the gastrointestinal tract and in part reddening of the lungs were observed. A dose of 50 mg/kg was tolerated without any clinical signs.

The following deaths were observed during the post-treatment phase:

Dose [mg/kg bw]

No of deaths

Time of death

Mortality rate [%]

males

50

0

-

0

100

0

-

0

200

1

4 h

20

250

4

4-8 h

80

500

4

1 h

80

800

5

2-4 h

100

females

50

0

-

0

100

1

1 h

20

200

1

1 d

20

250

4

4-8 h

80

310

5

4-8 h

100

The resulting oral LD50 for males and females together was calculated to be 216 mg/kg bw (CI 172 to 272 mg/kg bw) based on test material. This calculated to 124 mg/kg bw for the active ingredient.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
124 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Link to relevant study records

Referenceopen allclose all

Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of experimental phase: 21 June 2017 End of experimental phase: 12 July 2017 Study completion: 13 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Version / remarks:
adopted on 24 February 1987
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
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS Methods 870.1000 “Acute Toxicity Testing - Background”
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
standard acute method
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Species and strain: Rat, Hsd: Sprague Dawley SD
Sex: Males and females (nulliparous and non-pregnant)
Age at order: 6 to 8 weeks old (first batch used for 1000 mg/kg body weight)
6 to 7 weeks old (second batch used for 2000 mg/kg body weight)
Weight ordered: 176 to 200 grams (first batch used for 1000 mg/kg body weight)
150 to 174 grams (second batch used for 2000 mg/kg body weight)
Supplier: Envigo RMS srl, San Pietro al Natisone (UD), Italy
Breeder: Envigo RMS B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
Date of arrival: 14 June 2017 (both batches)
Weight range at arrival: 161.4 to 189.2 grams for males; 184.2 to 191.7 grams for females
(first batch used for 1000 mg/kg body weight)
168 to 171 grams for males; 159 to 168 grams for females
(second batch used for 2000 mg/kg body weight)
Acclimatisation period: At least 5 days
Veterinary health check: During acclimatisation period

ENVIRONMENTAL CONDITIONS
Animals per cage: Up to 5 of one sex during acclimatisation; individually caged during the study
Housing: Clear polysulfone H-Temp solid bottomed cages (Tecniplast Gazzada S.a.r.l., Buguggiate, VA, Italy) measuring 59.5×38×20 cm during acclimatisation period and 42.5×26.6×18.5 cm during the study with nesting material provided into suitable bedding bags.
Cage control: Daily inspected and changed as necessary (at least 3 times/week)
Water Drinking water supplied to each cage via a water bottle
Water supply: Ad libitum
Diet: 4 RF 18 (Mucedola S.r.l., Via G. Galilei, 4, 20019, SettimoMilanese (MI) Italy)
Diet supply: Ad libitum throughout the study
Room lighting: Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours
Air changes: Approximately 15 to 20 air changes per hour
Temperature range: 22 °C±2 °C
Relative humidity range: 55%±15%
Type of coverage:
semiocclusive
Details on dermal exposure:
TEST SITE
- Area of exposure: Dorsal surfaces of the trunk
- % coverage: Approximately 10% of body surface
- Type of wrap if used: Elastic adhesive bandage and gauze patch

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, with cotton wool soaked with lukewarmwater
- Time after start of exposure: 24 hours

Duration of exposure:
The animals remained in contact with the test item for 24 hours
Doses:
1000 and 2000 mg/kg body weight
No. of animals per sex per dose:
5 males anf 5 females at 1000 mg/kg body weight
5 males anf 5 females at 2000 mg/kg body weight
Control animals:
not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were observed for clinical signs
the day of dosing: on dosing, approximately 1 hour after dosing, approximately 2 hours after dosing and approximately 4 hours after dosing. Daily thereafter for a total of 14 days.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat. (total fraction)
Remarks on result:
no indication of skin irritation up to the relevant limit dose level
Mortality:
No mortality occurred
Clinical signs:
No signs of toxicity were observed in male or female animals during the observation period. Only blue staining in treated site, due to the colour of the substance, was observed in all male and female animals from Day 2 up to Day 15.
Body weight:
The body weight changes observed during the study were within the expected range for this species and age of animals.
Gross pathology:
Only blue staining on the treatment site, due to the colour of the substance, was found at necropsy in all animals at termination of the study.
Interpretation of results:
GHS criteria not met
Conclusions:
Basic Blue 159, has no toxic effect on the rat following dermal exposure over a 24 hour period at a level of 2000 mg/kg body weight. The LD50 is above 2000 mg/kg body weight.
Executive summary:

The acute toxicity of Basic Blue 159 was investigated following dermal administration of a single dose to the Sprague Dawley rat followed by a 14-day observation period. A single dose of 1000 mg/kg body weight was administered to a group of 5 male and 5 female animals for 24 hours. After 14 days, all animals were killed and subjected to necropsy to examine macroscopic changes. No mortality occurred and no signs of toxicity were observed in male or female animals during the observation period. A second group of 5 male and 5 female animals was then dosed at 2000 mg/kg body weight for 24 hours, which were also necropsied after 14 days of observation. No mortality occurred and no signs of toxicity were observed in male or female animals during the observation period. The body weight changes observed during the study were within the expected range for this species and age of animals. Only blue staining on the treatment site, due to the colour of the substance, was found at necropsy in all animals at termination of the study. These results indicate that the test item, Basic Blue 159, has no toxic effect on the rat following dermal exposure over a 24 hour period at a level of 2000 mg/kg body weight. The lack of mortality demonstrates the LD50 to be greater than 2000 mg/kg body weight. European Directives concerning the classification, packaging and labelling of dangerous substances (Council Regulation (EC)No. 1272/2008 and subsequent revisions) would indicate the following:

Classification No category

Signal word No signal word required

Hazard statement No hazard statement required

Endpoint:
acute toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Read-across
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
see attached justification document in section 13

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Basic Blue 159 trichlorozincate / CAS 93783-70-1 / EC 298-265-2
Target: Basic Blue 159 methyl sulfate / CAS 83969-12-4 / EC 281-589-3

3. ANALOGUE APPROACH JUSTIFICATION
see attached justification document in section 13

Reason / purpose for cross-reference:
read-across source
Test type:
standard acute method
Limit test:
no
Species:
rat
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat. (total fraction)
Remarks on result:
no indication of skin irritation up to the relevant limit dose level
Interpretation of results:
GHS criteria not met
Conclusions:
Basic Blue 159 trichlorozincate has no toxic effect on the rat following dermal exposure over a 24 hour period at a level of 2000 mg/kg body weight. The LD50 is above 2000 mg/kg body weight. The same is considered for the solid part of the target substance Basic Blue 159 methyl sulfate.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

An acute oral toxicity study was carried out with the test item in male and female rats. Five rats/sex/dose groups received a single gavage of the test item dissolved in distilled water at dose levels of 50, 100, 200, 250, 500, 800 mg/kg bw for males and 50, 100, 200, 250, 310 mg/kg bw for females. The animals were observed for signs of toxicity for 14 days thereafter.

Clinical signs observed in animals after a single administration of 100 to 800 mg/kg bw Basic Blue 159 were poor general condition, piloerection, sedation and prone or lateral position. Occasionally, tonic-clonic convulsions, laboured respiration and stilted gait occurred. A slight delay in body weight development was observed in females at a dose of 200 mg/kg bw in the first week of the study. In the pathological-anatomical examination of the deceased male and female rats at doses of 100 to 800 mg/kg bw, non-specific damage to the gastrointestinal tract and in part reddening of the lungs were observed. A dose of 50 mg/kg was tolerated without any clinical signs.

The following deaths were observed during the post-treatment phase:

Dose [mg/kg bw]

No of deaths

Time of death

Mortality rate [%]

males

50

0

-

0

100

0

-

0

200

1

4 h

20

250

4

4-8 h

80

500

4

1 h

80

800

5

2-4 h

100

females

50

0

-

0

100

1

1 h

20

200

1

1 d

20

250

4

4-8 h

80

310

5

4-8 h

100

The resulting oral LD50 for males and females together was calculated to be 216 mg/kg bw (CI 172 to 272 mg/kg bw) based on test material. This calculated to 124 mg/kg bw for the active ingredient.

The acute toxicity of Basic Blue 159 was investigated following dermal administration of a single dose to the Sprague Dawley rat followed by a 14-day observation period. A single dose of 1000 mg/kg body weight was administered to a group of 5 male and 5 female animals for 24 hours. After 14 days, all animals were killed and subjected to necropsy to examine macroscopic changes. No mortality occurred and no signs of toxicity were observed in male or female animals during the observation period. A second group of 5 male and 5 female animals was then dosed at 2000 mg/kg body weight for 24 hours, which were also necropsied after 14 days of observation. No mortality occurred and no signs of toxicity were observed in male or female animals during the observation period. The body weight changes observed during the study were within the expected range for this species and age of animals. Only blue staining on the treatment site, due to the colour of the substance, was found at necropsy in all animals at termination of the study. These results indicate that the test item, Basic Blue 159, has no toxic effect on the rat following dermal exposure over a 24 hour period at a level of 2000 mg/kg body weight. The lack of mortality demonstrates the LD50 to be greater than 2000 mg/kg body weight.

Justification for classification or non-classification

Based on the oral LD50 of 124 mg/kg bw a.i., the test item is classified as Category 3.