Registration Dossier

Administrative data

Description of key information

Oral: The acute oral LD50 was determined to be > 2000 mg/kg bw in rats. 
Dermal: The acute dermal LD50 was determined to be > 2000 mg/kg bw in rats.

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:
2003-09-03 - 2003-11-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 423) performed under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
adopted on 22nd March 1996
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 8 weeks old
- Body weight at study initiation: 306 ± 25 g (males), 220 ± 3 g (females)
- Housing: 3 animals/sex/cage in polycarbonate cages with stainless steel lid (48 cm x 27 cm
x 20 cm), each cage contained one to seven animals during the acclimation period
- Diet: A04 C pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum, except for an overnight fasting period prior to testing (free access to water)
- Water: Drinking water filtered by a FG Millipore membrane (0.22 micron), ad libitum
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 30 - 70%
- Air changes: approximately 12 air changes/hour
- Photoperiod: 12 h / 12 h

IN-LIFE DATES: From: 2003-09-11 To: 2003-10-02
Route of administration:
oral: gavage
Vehicle:
other: methylcellulose
Details on oral exposure:
VEHICLE:
- Concentration in vehicle: 20 or 200 mg/mL, based on dose volume
- Amount of vehicle/dose volume: 10 mL/kg bw
- Justification for choice of vehicle: to make dosing by gavage possible
- Purity: no data

DOSAGE PREPARATION:
- The test item was prepared in a 0.5% suspension of methylcellulose in purified water.

CLASS METHOD:
- Rationale for the selection of the starting dose: As no information on the toxic potential of the test item was available, for animal welfare
reasons, the starting dose of 200 mg/kg bw was chosen.
Doses:
200 and 2000 mg/kg bw (tested concentrations)
No. of animals per sex per dose:
3 males (200 mg/kg)
3 males/ 3 females (2000 mg/kg)
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days

- Mortality/Viability and frequency of observations (clinical signs): The animals were observed frequently during the hours following administration of the test item, for detection of possible treatment-related clinical signs. Thereafter, observation of the animals was made at least once a day. Type, time of onset and duration of clinical signs were recorded for each animal individually.

- Frequency of weighing: The animals were weighed individually just before administration of the test item on day 1 and then on days 8 and 15. The body weight gain of the treated animals was compared to that of CIT control animals with the same initial body weight.

- Necropsy of survivors performed: yes, on day 15, all animals were killed by carbon dioxide asphyxiation and examined macroscopically. All study animals were subjected to a macroscopic examination as soon as possible after death. After opening the thoracic and abdominal cavities, a macroscopic examination of the main organs (digestive tract, heart, kidneys, liver, lungs, pancreas, spleen and any other organs with obvious abnormalities) was performed. No organ samples were taken.
Statistics:
No statistical testing was performed.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was observed at any dose level tested.
Clinical signs:
No clinical signs were observed in the animals given 200 mg/kg bw. At the 2000 mg/kg bw dose-level, piloerection and dyspnea, together with hypoactivity in females, were observed in all animals on day 1.
Body weight:
When compared to CIT historical control animals, a slightly reduced body weight gain was recorded in 2/3 males given 200 mg/kg bw during the first or second week of the study. At 2000 mg/kg bw, a reduced body weight gain was noted in 1/3 females during the second week of the study. The overall body weight gain of the other animals was not affected by the treatment with the test item.
Gross pathology:
Macroscopic examination revealed no apparent abnormalities.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
Value:
2 000 mg/kg bw

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:
2003-09-03 - 2003-11-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 402) performed under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Version / remarks:
adopted on 24th February 1987
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Version / remarks:
adopted on 31st July 1992
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 8 weeks old
- Body weight at study initiation: 309 ± 5 g (males), 231 ± 5 g (females)
- Housing: animals were housed individually in polycarbonate cages with stainless steel lid (35.5 cm x 23.5 cm x 19.3 cm), each cage contained autoclaved sawdust (SICSA, Alfortville, France).
- Diet: A04 C pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum
- Water: Drinking water filtered by a FG Millipore membrane (0.22 micron), ad libitum
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 30 - 70%
- Air changes: approximately 12 air changes/hour
- Photoperiod: 12 h / 12 h

IN-LIFE DATES: From: 2003-09-17 To: 2003-10-01
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: dorsal area of each animal was clipped (males: appr. 5 x 7 cm; females: appr. 5 x 6 cm)
- Coverage: Approximately 10 % of the total body surface
- On test day 1 a single dose of 2000 mg/kg bw of the test item was placed on a hydrophilic gauze pad (pre-moistened with 2 mL of purified water) and then applied on the intact skin with an aerated semi-occlusive dressing
- Type of wrap: Restraining bandage, this dressing prevented ingestion of the test item by the animal

REMOVAL OF TEST SUBSTANCE
- Washing: Any residual test item was removed using a moistened cotton pad
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount applied: 2000 mg/kg bw
- Constant volume

VEHICLE
- Amount applied: 2 mL per rat
Duration of exposure:
24 h
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5 rats
Control animals:
other: not applicable
Details on study design:
- Duration of observation period following administration: 14 days

- Mortality/Viability and frequency of observations (clinical signs): The animals were observed frequently during the hours following administration of the test item, for detection of possible treatment-related clinical signs. Thereafter, observation of the animals was made at least once a day until termination. Type, time of onset and duration of clinical signs were recorded for each animal individually.

- Frequency of weighing: The animals were weighed individually just before administration of the test item on day 1 and then on days 8 and 15. The body weight gain of the treated animals was compared to that of CIT control animals with a similar initial body weight.

- Necropsy of survivors performed: At the end of the observation period, all animals were killed by carbon dioxide asphyxiation and examined macroscopically. All study animals were subjected to a macroscopic examination as soon as possible after death. After opening the thoracic and abdominal cavities, a macroscopic examination of the main organs (digestive tract, heart, kidneys, liver, lungs, pancreas, spleen and any other organs with obvious abnormalities) was performed. No organ samples were taken.
Statistics:
No statistical analysis was used.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality was observed at 2000 mg/kg bw.
Clinical signs:
Signs of toxicity related to dose levels: None.
A white coloration of the skin was noted in all animals on day 2.
Body weight:
The overall body weight gain of the treated animals was similar to that of historical control animals.
Gross pathology:
Macroscopic examination of the main organs of the animals revealed no apparent abnormalities
Other findings:
Signs of toxicity (local): None.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
Value:
2 000 mg/kg bw

Additional information

Acute oral toxicity:

In the key oral toxicity study (CIT 26394, 2003, OECD 423) 3 male (200 mg/kg bw), 3 male and female (2000 mg/kg bw) fasted Sprague Dawley rats were exposed each by gavage to non-micronized ETH50. Animals were observed for 14 days. There was no mortality. No clinical signs and no mortality were observed in the animals given 200 mg/kg bw. At the 2000 mg/kg bw dose-level, piloerection and dyspnea, together with hypoactivity in females, were observed in all animals on day 1. A slight reduced body weight gain was recorded in 2/3 males given 200 mg/kg bw during the first or second week of the study compared to historical controls. At 2000 mg/kg bw, a reduced body weight gain was noted in 1/3 females during the second week of the study. The overall body weight gain of the other animals was not affected by the treatment with the test item. At necropsy, no apparent macroscopic abnormalities were observed. The oral LD50 was determined to be > 2000 mg/kg bw.

The test substance used in the key study represents the form of the substance registered (i.e ETH50 as pure (bulk) non-nano material).  As such, however, it is not usable for its intended use in cosmetic products. In order to transform it into suitable cosmetic ingredient, ETH50 has to be micronized in a water-mill. As a consequence of the extreme hydrophobicity of substance, surfactants need to be added to introduce it in the water phase. After milling, rheology modifiers prevent the nano-particles from agglomerating and stabilize the resulting preparation. Thus, the substance is marketed in a 50 % mixture together with different components; it is not available as pure material in nanoform.

Thus, a test substance formulation relating to the marketed mixture has been tested in addition. In this supporting study with a 49.5 % formulation of micronized ETH50 (nano-form), the oral LD50 was higher than 2000 mg/kg bw in rats (CIT 32808, 2007 ; OECD 423).

 

Acute inhalation toxicity:

An OECD-guideline and GLP compliant inhalation (nose only) toxicity study (RCC B38698, 2007) on a formulation containing micronized ETH50 (relating to the marketed mixture for the use in cosmetics), diluted in water (final concentration 10% ETH50) indicated an LC50 of > 4976 mg/m³ when administered to rats for a single 4 -hour period. These findings confirm the absence of any acute inhalative toxicity of ETH50. Since ETH50 has not been tested up to the limit concentration, no final conclusion on classification can be drawn, although in light of the present data from oral and dermal toxicity studies, acute toxicity via the inhalative route is unlikely. Furthermore, inhalative exposure is not considered to be the major route of exposure. Therefore, an acute toxicity study via the inhalative route is scientifically not required in accordance with column 2 of REACH Annex VIII, and is not in line with animal welfare requirements.

 

Acute dermal toxicity:

In an acute dermal toxicity study (CIT 26395, 2003, OECD 402, acc. to GLP), young adult Sprague Dawley rats (5 males and 5 females) were dermally exposed to a single dose of 2000 mg/kg bw non-micronized ETH50 to the clipped skin (dorsal and dorso-lateral parts of the trunk) and covered by semi-occlusive dressing for 24 hours. The animals were observed for 14 days. No mortality occurred and no signs of systemic toxicity or skin effects were seen. No macroscopic pathologic abnormalities were noted. Accordingly, the LD 50 was determined to be > 2000 mg/kg bw.


Justification for classification or non-classification

The present data do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008, and therefore, a non-classification is warranted.