Registration Dossier

Administrative data

Description of key information

Not harmful/toxic if swallowed

Not harmful/toxic in contact with skin

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 September 27 to October 18t, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Version / remarks:
adopted February 24t, 1987
Qualifier:
according to
Guideline:
EU Method B.1 (Acute Toxicity (Oral))
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd.
- Age at study initiation: 8 weeks males; 10 weeks females.
- Weight at study initiation: 191 - 206 g males; 168 - 181 g females.
- Fasting period before study: 12 - 18 hours before test administration. Access to water was not interrupted. 2-3 hours after dosing food was again presented.
- Housing: groups of five in Makrolon type-3 cages with standard softwood bedding.
- Diet: pelleted standard Kliba 343, rat maintenance diet, ad libitum.
- Water: community tap water from Itingen, ad libitum.
- Acclimation: one week under laboratory conditions, after veterinary examination.

HUSBANDRY CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 10 - 15 air changes per hour.
- Photoperiod: 12hrs dark / 12 hrs artifical fluorescent light.
- Other: room was airconditioned, music during the light period.
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
bi-distilled
Details on oral exposure:
APPLICATION VOLUME/Kg bw
10 ml at 2000 mg/kg

PREPARATION
The test article was placed into a glass beaker on a tared balance and the vehicle was added. A weight by volume dilution was prepared using a homogenizer. Homogeneity of the test article in the vehicle maintained during treatment using a magentic stirrer.
The preparation was made immediately prior to dosing.
Doses:
10 ml at 2000 mg/kg b.w.
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Details on study design:
- Duration of observation period following administration: 15 days.
- Clinical signs observed: each animal was examined for changes in appearance and behaviour four times during day 1, and daily during days 2-15. All abnormalities were recorded. The animals were checked for the following clinical signs: aggressiveness, vocalization, restlessness / excitation, nervousness, fear, sedation, somnolence, sleep, coma, rhinorrhea, epistaxis, apnea, dyspnea, rales, chromodacryorrhea, exophthalmos, miosis, mydriasis, whitish discharge, lid adhesion, lacrimation, negative corneal reflex, akinesia, ataxia, dropped head, hyperkinesia, hypokinesia, paralysis, flaccid, paralysis, spastic, paddling movements, stiff gait, rolling movements, ventral body position, latero-abdominal position, hunched posture, spasms, tonic muscle spasms, clonic muscle spasms, opisthotonus, saltatory, spasms, trismus, tremor, muscle-twitching emaciation and muscle-twitching generalized, erythema, edema, necrosis, loss of weight, diarrhea, ruffled fur, salivation, pallor, cyanosis.
- Frequency of observations: mortality/viability were recorded four times during test day 1(at 1, 2, 3 and 5 hours) and daily during days 2 - 15.
- Frequency of weighing: test day 1 (pre-administration), day 8 and 15.
- Necropsy: necropsies were performed by experienced prosectors. All animals were necropsied. All the animals which survived until the scheduled termination were euthanized by intraperitoneal injection of sodium pentobarbitone.
Statistics:
The LOGIT-Model could not be applied to the observed rates of death. The toxicity was estimated without the use of a statistical model.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality occurred up to 15 days post-treatment.
Clinical signs:
2000 mg/kg: males/females exhibited ruffled fur, diarrhea, hunched posture. Males exhibited sedated.
All animals had recovered after 10 observation days.
Body weight:
The body weight gain of the animals was not affected by the test article treatment throughout the entire study period.
Gross pathology:
No obvious macroscopical organ findings were seen in any animal.

Clinical signs

Summary

Test day

1 1 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time after treatment (hrs) 1 2 3 5

Males

Behaviour sedated - - - x - - - - - - - - - - - - - -
Hunched posture x x x x x x x x x - - - - - - - - -
Ruffled fur x x x x x x x x x x x x x - - - - -
Diarrhea - x x x x - - - - - - - - - - - - -

Females

Hunched posture - - - x - - - - - - - - - - - - - -
Ruffled fur - x x x x x x x x x x x x - - - - -
Diarrhea - x x x x - - - - - - - - - - - - -

X = sign observed

- = observation performed, sign not observed

Test day 1 1 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Time after treatment (hrs) 1 2 3 5
Animal N. Sign (G)
1M Ruffled fur (3) - 1 1 1 1 1 1 1 1 1 - - - - - - - -
Diarrhea (1) - 1 1 - - - - - - - - - - - - - - -
2M Ruffled fur (3) - 1 1 1 1 1 1 1 1 1 1 - - - - - - -
Diarrhea (1) - 1 1 1 1 - - - - - - - - - - - - -
3M Ruffled fur (3) - 1 1 1 1 1 1 1 1 1 1 1 1 1 - - - -
Diarrhea (1) - 1 1 1 1 - - - - - - - - - - - - -
4M Behaviour sedated (3) - - - 1 - - - - - - - - - - - - - -
Hunched posture (1) 1 1 1 1 1 1 1 1 1 - - - - - - - - -
Ruffled fur (3) 1 1 1 1 1 1 1 1 1 1 - - - - - - - -
Diarrhea (1) - 1 1 1 1 - - - - - - - - - - - - -
5M Ruffled fur (3) - 1 1 1 - - - - - - - - - - - - - -
Diarrhea (1) - 1 1 - - - - - - - - - - - - - - -
6F Ruffled fur (3) - 1 1 1 1 1 1 1 1 1 1 - - - - - - -
Diarrhea (1) - 1 1 1 1 - - - - - - - - - - - - -
7F Ruffled fur (3) - 1 1 1 1 1 1 1 1 1 - - - - - - - -
Diarrhea (1) - 1 1 - - - - - - - - - - - - - - -
8F Ruffled fur (3) - 1 1 1 1 1 1 1 1 1 1 1 1 1 - - - -
Diarrhea (1) - 1 1 1 1 - - - - - - - - - - - - -
9F Hunched posture (1) - - - 1 - - - - - - - - - - - - - -
Ruffled fur (3) - 1 1 1 1 1 1 1 1 1 - - - - - - - -
Diarrhea (1) - 1 1 1 1 - - - - - - - - - - - - -
10F Ruffled fur (3) - 1 1 1 1 1 1 1 1 1 1 - - - - - - -
Diarrhea (1) - 1 1 1 1 - - - - - - - - - - - - -

- = observation performed, sign not observed

(G) = max. grade

Interpretation of results:
other: not classified, according to the CLP Regulation (EC) No 1272/2008
Conclusions:
LD50 (males and female) > 2000 mg/kg bw
Executive summary:

The substance was assessed for its acute toxicity to rats in a limit test, according to the OECD guideline 401 and the EU Method B.1. The acqueous solution of the substance was administrated by oral gavage to five female and five male Wistar rats in a concentration of 2000 mg/kg bw. The rats were observed for mortality, clinical signs, macroscopic changes and vitality four times during test day 1 (at 1, 2, 3 and 5 hrs) and daily during days 2 - 15. The animals were weighed on test day 1 (pre-administration), day 8 and 15.

During the observation period, no mortality was observed and no macroscopical findings were noted. The body weight was increased. Clinical signs observed in some animals were sedated behaviour and hunched posture, while ruffled fur and diarrhea was observed in all animals until day 10 and day 2 respectively. No obvious macroscopical organ findings were seen in any animal.

Conclusion

LD50 (males and female) > 2000 mg/kg bw

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 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
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 27 to October 18, 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Version / remarks:
adopted February 24th, 1987
Qualifier:
according to
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd.
- Age at study initiation: 10 weeks males; 12 weeks females.
- Weight at study initiation: 211 - 243 g males; 189 - 205 g females.
- Housing: individually in Makrolon type-2 cages with standard softwood bedding.
- Diet: pelleted standard Kliba 343, rat maintenance diet, ad libitum.
- Water: community tap water from Itingen, ad libitum.
- Acclimation: one week under laboratory conditions and after veterinary examination.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 40 - 70 %
- Air changes: 10-15 air changes per hour.
- Photoperiod: 12 hrs dark / 12 hrs artificial fluorescent light.
- Other: music during the light period, room was air-conditioned.
Type of coverage:
semiocclusive
Vehicle:
water
Details on dermal exposure:
TEST SITE
- Preparation of site of exposure: approximately 24 hours before treatment, the backs of the animals were clipped with an electric clipper.
- % coverage: appr. 10 % of the total body surface.
- Application of test material: applied evenly on the skin with a syringe.
- Type of wrap if used: semi - occlusive dressing wrapped around the abdomen and fixed with an elastic adhesive bandage.

TEST MATERIAL
- Amount volume: 4 ml at 2000 mg/kg
- Preparation of test sample: the test article was placed into a glass beaker on a tared balance and the vehicle (bi-distilled water) was added. A weight/volume dilution was prepared using a homogenizer. Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer. The preparation was made immediately prior to dosing.

REMOVAL OF TEST SUBSTANCE
- Washing: with lukewarm tap water. The skin was then dried with disposable paper towels.
- Time after start of exposure: 24 hours after the application.
Duration of exposure:
24 hours.
Doses:
2000 mg/kg bw.
No. of animals per sex per dose:
5 male and 5 females.
Control animals:
no
Details on study design:
- Duration of observation period following administration: 15 days.
- Frequency of observations: mortality and viability were recorded four times during test day 1 and daily during days 2 - 1 5.
- Frequency of weighing: on the first day (pre-administration), at day 8 and day 15.
- Clinical signs observed: each animal had an examination for changes in appearance and behaviour four times during day 1 and daily during days 2-15. All abnormalities were recorded. Due to the 24 hour semi-occlusive treatment, the local findings were observed starting with day 2 of test. The animals were checked for the following clinical signs: aggressiveness, vocalization, restlessness / excitation, nervousness, fear, sedation, somnolence, sleep, coma, rhinorrhea, epistaxis, apnea, dyspnea, rales, chromodacryorrhea, exophthalmos, miosis, mydriasis, whitish discharge, lid adhesion, negative corneal reflex, akinesia, ataxia, dropped head, hyperkinesia, hypokinesia, paralysis flaccid, paralysis spastic, paddling movements, stiff gait, rolling movements, ventral body position, latero-abdominal position, hunched posture, spasms, tonic muscle spasms, clonic muscle spasms, opisthotonus, saltatory spasms, trismus, tremor, muscle-twitching generalized, erythema, edema, necrosis, crusts, scale formations, loss of weight, diarrhea, emaciation, ruffled fur, necrosis of tissue of application area, salivation, pallor, cyanosis.
- Necropsy: all animals were necropsied. The surviving animals were euthanised at the end of the observation period by intraperitoneal injection of sodium pentobarbitone.
Statistics:
The LOGIT-Model could not be applied to the observed rate of death. The toxicity was estimated without the use of a statistical model.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality observed.
Clinical signs:
The rats exhibited blackened skin throughout the entire study period. No systemic signs were observed in any animal.
Body weight:
The body weight gain of the animals was not affected throughout the study by test article treatment.
Gross pathology:
No obvious macroscopical organ findings noted.

Clinical signs

Summary

Test day

1 1 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time after treatment (hrs)

1 2 3 5
Skin black (back) - Males - - - - X X X X X X X X X X X X X X
Skin black (back) - Females - - - - X X X X X X X X X X X X X X

X = sign observed

- = observation performed, sign not observed

Test day

1 1 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Time after treatment (hrs)

1 2 3 5
Anima N.  Sign (G)
1M Skin black (back) 1 - - - - 1 1 1 1 1 1 1 1 1 1 1 1 1 1
2M Skin black (back) 1 - - - - 1 1 1 1 1 1 1 - - - - - - -
3M Skin black (back) 1 - - - - 1 1 1 1 1 1 1 1 1 1 1 1 1 1
4M Skin black (back) 1 - - - - 1 1 1 1 1 1 1 - - - - - - -
5M Skin black (back) 1 - - - - 1 1 1 1 1 1 1 1 1 1 1 - - -
6F Skin black (back) 1 - - - - 1 1 1 1 1 1 1 1 1 1 1 1 1 1
7F Skin black (back) 1 - - - - 1 1 1 1 1 1 1 1 1 1 1 1 1 1
8F Skin black (back) 1 - - - - 1 1 1 1 - - - - - - - - - -
9F Skin black (back) 1 - - - - 1 1 1 1 - - - - - - - - - -
10F Skin black (back) 1 - - - - 1 1 1 1 1 1 1 - - - - - - -

- = observation performed, sign not observed

(G) = max. grade

Interpretation of results:
other: not classified, according to the CLP Regulation (EC) No 1272/2008
Conclusions:
LD50 (males and female) > 2000 mg/kg bw
Executive summary:

The purpose of the dermal toxicity study was to assess the toxicological profile of test item when administered to rats by a single semi-occlusive dermal application, with an observation period of 15 days. The test was perfromed accodring to the OECD guideline 402. The test article was applied to the skin of rats of both sexes for 24 hours at a single dose of 2000 mg/kg bw. The substance was removed after 24 hours exposure and the skin was washed. All the animals were subjected to necropsy at the end of the observation period.

No dead occurred during the experiment and no macroscopic findings were noted. All animals, showed a black-coloured back, from the 2nd day until the end of the observation period, but no mortality occured.

Conclusion

LD50 (males and female) > 2000 mg/kg bw

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 µg/kg bw

Additional information

ACUTE TOXICITY - ORAL ROUTE

Three tests are available for the assessment of the potential oral acute toxicity of Direct Black 168. In all cases, a single dose of 2000 mg/kg bw of the substance was administered to rats; rats were observed for mortality, clinical signs and vitality for 14/15 days after exposure and necropsy was performed after the observation period.

In the key study, no mortality occurred up to 15 days post-treatment. Males and females exhibited ruffled fur, diarrhea, hunched posture. Additionally, males appeared sedated. All animals had recovered after 10 observation days; the body weight gain was not impacted and no macroscopical findings were seen in any animal. The tested substance was characterized by an appreciable content of the main component and it can be considered as representative for the substance under registration.

In the study performed in 2000 one male died after 6 hours; the necropsy of the male that died after 6 hours did not revealed any significant changes. All the other animals survived (i.e. 2 males and 3 females). 10 minutes after exposure, a clear decrease in motor activity was observed in all animals, with postration, piloerection and respiratory distress. 24 hours after administration, there was a notable recovery of symptoms, except for prostration and piloerection. At 48 hours, the symptoms were recovered and the animals appeared normal and survived until the 14th day. No macroscopic changes found after the necropsy of the survived animals. As in the previously described experiment, the tested substance can be considered as representative for the substance under registration.

In the third study, the tested substance was characterized by a lower main component content respect to the other tests. None of the treated animals died. All the animals showed a soft, blackish faeces during the first three days following administration. The body weight gain was not impacted and in the necropsies performed only one male demonstrated a slightly larger right kidney which also had a dilated renal pelvis.

In conclusion, the studies outcomes let to identify the LD50, for both males and females, as higher than 2000 mg/kg bw.

 

ACUTE TOXICITY - INHALATION ROUTE

No acute toxicity study by inhalation route is available on Direct Black 168.

Nevertheless, because of the physical state and properties of the substance inhalation is not an appropriate route of exposure. Particle size distribution showed that the median mass distribution diameter of Direct Black 168 resulted to be 13.8 µm; particles having a diameter lower than 4 µm are ca 2 %. Thus, most of the Direct Black 168 particles are expected to remain in the upper respiratory tract, which is characterized by efficacious defence mechanisms able to remove them; therefore, respirable particles, able to enter the deepest part of the lung, the non-ciliated alveoli, can be considered a minimal portion. This consideration, together with the consideration that the substance is manufactured and handled with suitable risk management measures and with the suitable personal protective equipments, lets to consider the possible absorption of the substance by inhalation route as negligible.

 

ACUTE TOXICITY - DERMAL ROUTE

Direct Black 168 was assayed for acute dermal potential toxicity; the experiment was conducted according to the OECD guideline 402. Test item was administered to rats by a single semi-occlusive dermal application at the concentration of 2000 mg/kg bw, with an observation period of 15 days; all the animals were subjected to necropsy at the end of the observation period.

No dead occurred during the experiment and no macroscopic findings were noted. All animals, showed a black-coloured back, from the 2nd day until the end of the observation period, but no mortality occurred.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, 3.1 acute toxicity section, acute toxicity means those adverse effects occurring following oral or dermal administration of a single dose of a substance or a mixture, or multiple doses given within 24 hours, or an inhalation exposure of 4 hours. Substances can be allocated to one of four toxicity categories based on acute toxicity by the oral, dermal or inhalation route according to the numeric criteria. Acute toxicity values are expressed as (approximate) LD50 (oral, dermal) or LC50 (inhalation) values or as acute toxicity estimates (ATE).

The oral LD50 value was established to be greater than 2000 mg/kg body weight, therefore the test substance is out of any classification limit for acute oral toxicity (oral acute toxicity category 4: 300 < ATE ≤ 2000 mg/kg bw).

Dermal LD50 value was established to be greater than 2000 mg/kg body weight, therefore the test substance is out of any classification limit for acute dermal toxicity (dermal acute toxicity category 4: 1000 < ATE ≤ 2000 mg/kg bw).

Inhalation exposure is unlikely; no acute toxicity value is available and no further investigation is required.

In conclusion, the substance is not classified for oral, nor dermal acute toxicity, according to the CLP Regulation (EC) No 1272/2008.