Registration Dossier

Administrative data

Description of key information

Based on the results of an acute oral toxicity study performed in accordance with OECD 423, the substance needs to be classified as harmful if swallowed, as the LD50 cut-off value was considered to be 1000 mg/kg body weight. Based on the results of an acute dermal toxicity study, the substance does not need to be classified for acute dermal toxicity, as the LD50 was determined to exceed 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:
06 Jun 2017 - 04 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
December 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
December 2002
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF Guidelines
Version / remarks:
2000, including the most recent revisions
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
other: Crl:WI (Han)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 8-9 weeks old).
- Weight at study initiation: Body weight variation was 152gr to 186gr.
- Fasting period before study: Animals were deprived of food overnight (for a maximum of 20 hours).
- Housing: Group housing of 3 animals per cage in labeled Macrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 – 22
- Humidity (%): 45 - 68
- Air changes (per hr): ten or greater.
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 13 June 2017 to 04 July 2017
Route of administration:
oral: gavage
Details on oral exposure:
GAVAGE METHOD: plastic feeding tubes.

Frequency: single dosage, on Day 1.

VEHICLE:
Trial preparations were performed at the Test Facility to select the suitable vehicle and to establish a suitable formulation procedure.
Propylene glycol was the vehicle of choice.
Specific gravity: 1.036


MAXIMUM DOSE VOLUME APPLIED:
(10 mL/kg) body weight.


DOSAGE PREPARATION: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle and test item. No correction was made for the purity/composition of the test item.
Any residual volumes were discarded.

Doses:
2000 mg/kg body weight
300 mg/kg body weight


No. of animals per sex per dose:
2000 mg/kg body weight: 3
300 mg/kg body weight: 6 (2 groups of three females in a stepwise manner)
Control animals:
no
Details on study design:
The toxicity of the test item was assessed by stepwise treatment of groups of 3 females. The absence or presence of mortality of animals dosed at one step determined the next step, based on the test procedure defined in the guidelines. The onset, duration and severity of the signs of toxicity were taken into account for determination of the time interval between the dose groups. The first group was treated at a dose level of 2000 mg/kg body weight. Based on the results, two additional groups were dosed at 300 mg/kg body weight.

Animals were deprived of food overnight prior to dosing and until 3-4 hours after administration of the test substance. Water was available ad libitum.

- Duration of observation period following administration: 14 days.
- Frequency of observations: Postdose observations were performed at periodic intervals on the day of dosing (at least three times) and once daily thereafter.
Mortality/Viability: twice daily.
Body weights: Days 1 (pre-administration), 8 and 15.
Clinical signs: At periodic intervals on the day of dosing (Day 1) and once daily thereafter, until Day 15.
- Necropsy of survivors performed: All moribund animals and animals surviving to the end of the observation period were sacrificed by oxygen/carbon dioxide procedure. All animals assigned to the study were subjected to necropsy and descriptions of all internal macroscopic abnormalities were recorded.
- Other examinations performed: none.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 300 - < 2 000 mg/kg bw
Based on:
test mat.
Mortality:
At 2000 mg/kg body weight, two animals were sacrificed for humane reasons on Day 1.
At 300 mg/kg body weight, no mortality occurred.
Clinical signs:
At 2000 mg/kg body weight, comatose state, lethargy, flat posture, abnormal posture, lateral recumbency,
uncoordinated movements, hunched posture, rales, piloerection, watery discharge from the
eyes, ptosis, brown coloration of urine and/or hypothermia were noted for the animals on Day
1. Additionally, the surviving animal showed hunched posture and piloerection on Days 2
and/or 3.
At 300 mg/kg body weight, hunched posture, piloerection and/or ptosis were noted for all animals on Day 1 only.
Body weight:
The body weight gain shown by the surviving animals over the study period was considered to be similar to that expected for normal untreated animals of the same age and strain.
Gross pathology:
At 2000 mg/kg body weight, gelatinous contents of the gastrointestinal tract (stomach, duodenum and/or jejunum) and reddish contents of the urinary bladder were found in the animals sacrificed for humane reasons during the study, at macroscopic post mortem examination. No abnormalities were found in the remaining animal. At 300 mg/kg body weight, no abnormalities were found at macroscopic examination
Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The oral LD50 value of ethanol, 2,2’-[(3-methylphenyl)imino]bis- in Wistar rats was established to be within the range of 300-2000 mg/kg body weight.
According to CLP and GHS regulations the subtstance is classified as category 4, labelled with H302, (harmful if swallowed).
Executive summary:

An assessment of acute oral toxicity with ethanol, 2,2’-[(3-methylphenyl)imino]bis- in the rat (Acute Toxic Class Method) was performed according to OECD/EC test guidelines and GLP principles.The test item was administered by oral gavage to one groups three female Wistar rats at 2000 mg/kg body weight. Two animals were sacrificed for humane reasons on Day 1. At day 1 comatose state, lethargy, flat posture, abnormal posture, lateral recumbency, uncoordinated movements, hunched posture, rales, piloerection, watery discharge from the eyes, ptosis, brown coloration of urine and/or hypothermia were noted for the animals. Additionally, the surviving animal showed hunched posture and piloerection on Days 2 and/or 3. Based on the results, two additional groups were dosed at 300 mg/kg body weight. At 300 mg/kg body weight, no mortality occurred but hunched posture, piloerection and/or ptosis were noted for all animals on Day 1.

The body weight gain shown by the animals over the study period was considered to be similar to that expected for normal untreated animals of the same age and strain.

At 2000 mg/kg body weight, gelatinous contents of the gastrointestinal tract (stomach, duodenum and/or jejunum) and reddish contents of the urinary bladder were found in the animals sacrificed for humane reasons during the study, at macroscopic post mortem examination. No abnormalities were found in the remaining animal. At 300 mg/kg body weight, no abnormalities were found at macroscopic examination.

The oral LD50 value of Ethanol, 2,2’-[(3-methylphenyl)imino]bis- in Wistar rats was therefore established to be within the range of 300-2000 mg/kg body weight.

According to the OECD 423 test guideline, the LD50 cut-off value was considered to be 1000 mg/kg body weight.

Based on these results, Ethanol, 2,2’[(3-methylphenyl)imino]bis- is classified according to CLP and GHS regulations the subtstance is classified as category 4, labelled with H302 , (harmful if swallowed).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
1 000 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:
19 March 2018 - 23 April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes
Test type:
fixed dose procedure
Limit test:
yes
Specific details on test material used for the study:
Stability in vehicle: The test item is stable in a solution of Propylene glycol for at least 5 hours at room temperature under normal laboratory conditions, for at least 6 days in the refrigerator and, for at least 3 weeks in the freezer (≤ -15°C) over the concentration range 1 to 200 mg/mL.

The test item was heated to approximately 80°C for 15 minutes approximately 2 hours before weighing.

Adjustment was made for specific gravity of the vehicle (1.036).
Species:
rat
Strain:
other: Crl: WI(Han)
Remarks:
Outbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant:yes
- Age at study initiation: approx. 9 - 12 weeks old
- Weight at study initiation: 166-212 g.
- Housing: Group housing (up to 5 animals of the same sex together) on arrival and individual housing during the study. Polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust were used during the study. For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom), except when interrupted by study procedures/activities.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum throughout the study, except during designated procedures.
- Water: Municipal tap-water, ad libitum
- Acclimation period: 5 days

The feed was analyzed by the supplier for nutritional components and environmental contaminants. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.
Periodic analysis of the water was performed, and it is considered that there were no known contaminants in the water that would interfere with the objectives of the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21
- Humidity (%): 30-51
- Air changes (per hr): >= 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 20 March 2018 To: 23 April 2018
Type of coverage:
semiocclusive
Vehicle:
propylene glycol
Remarks:
Specific gravity: 1.036
Details on dermal exposure:
RANGEFINDING STUDY
A range finding study was performed in order to select the dose causing no mortality or significant toxicity to be used in the main study. Initially, one animal was dosed at 1000 mg/kg. A period of at least 48 hours was allowed between the dosing of each animal. Based on the results one additional animal was dosed at 2000 mg/kg. Based on the results of the range finding study, in the main study two animals were dosed at 2000 mg/kg.

TEST SITE
- Area of exposure: 18 cm^2
- % coverage: approx. 10% of the total body surface
- Type of wrap if used: The test item was held in contact with the skin with a dressing, consisting of a surgical gauze patch (Surgy 1D), successively covered with Coban elastic bandage. A piece of Micropore tape was additionally used for fixation of the bandages/

REMOVAL OF TEST SUBSTANCE
- Washing (if done): with water
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount applied: 1000 & 2000 mg/kg bodyweight
- Constant volume or concentration used: yes, 10 mL/kg bodyweight
Duration of exposure:
24 hours
Doses:
1000 mg/kg bodyweight
2000 mg/kg bodyweight
No. of animals per sex per dose:
1 female: 1000 mg/kg bodyweight
3 females: 2000 mg/kg bodyweight
Control animals:
not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations: Mortality: twice daily; Clinical observations: at periodic intervals on the day of dosing (at least three times) and once daily thereafter; Body weights: on day 1 (pre-administration), day 8 and day 15.
- Necropsy of survivors performed: yes

The skin reactions were assessed approximately 24, 48 and 72 hours after the removal of the dressing and test item. Adjacent areas of untreated skin of each animal served as controls.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality occurred.
Clinical signs:
Red discoloration of the nose and ptosis were noted for one animal on Day 1.
Body weight:
Body weight loss was noted for one animal at Day 8 and body weight loss or reduced body weight gain was noted for the other two animals at Day 15.
Gross pathology:
No abnormalities were found at macroscopic post mortem examination of the animals.
Other findings:
Irritation and local effects:
No erythema or edema were scored for any of the animals at any time point. Fissures, scales, scabs and/or red discoloration were seen in the treated skin-area of the animals between Days 2 and 15. Scabs were noted on the back of one animal between Days 12 and 15.
Interpretation of results:
GHS criteria not met
Remarks:
Not classified according to Regulation (EC) No. 1272/2008.
Conclusions:
The dermal LD50 value of Ethanol, 2,2’-[(3-methylphenyl)imino]bis- in Wistar rats was determined to exceed 2000 mg/kg body weight. Based on this result, Ethanol, 2,2’-[(3-methylphenyl)imino]bis- is not classified according to GHS and CLP criteria.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw

Additional information

Oral

An assessment of acute oral toxicity with ethanol, 2,2’-[(3-methylphenyl)imino]bis-in the rat (Acute Toxic Class Method) was performed according to OECD/EC test guidelines and GLP principles. The test item was administered by oral gavage to one group of three female Wistar rats at 2000 mg/kg body weight. Two animals were sacrificed for humane reasons on Day 1. At day 1 comatose state, lethargy, flat posture, abnormal posture, lateral recumbency, uncoordinated movements, hunched posture, rales, piloerection, watery discharge from the eyes, ptosis, brown coloration of urine and/or hypothermia were noted for the animals. Additionally, the surviving animal showed hunched posture and piloerection on Days 2 and/or 3. Based on the results, two additional groups were dosed at 300 mg/kgbody weight. At 300 mg/kgbody weight, no mortality occurred but hunched posture, piloerection and/or ptosis were noted for all animals on Day 1. The body weight gain shown by the animals over the study period was considered to be similar to that expected for normal untreated animals of the same age and strain. At 2000 mg/kg body weight, gelatinous contents of the gastrointestinal tract (stomach, duodenum and/or jejunum) and reddish contents of the urinary bladder were found in the animals sacrificed for humane reasons during the study, at macroscopic post mortem examination. No abnormalities were found in the remaining animal. At 300 mg/kg body weight, no abnormalities were found at macroscopic examination. The oral LD50 value of Ethanol, 2,2’-[(3-methylphenyl)imino]bis- in Wistar rats was therefore established to be within the range of 300-2000 mg/kg body weight. According to the OECD 423 test guideline, the LD50 cut-off value was considered to be 1000 mg/kg body weight.

Dermal

The acute dermal toxicity of the substance was investigated in accordance with OECD 402 and according to GLP principles. A range finding study was performed in order to select the dose causing no mortality or significant toxicity to be used in the main study. Initially, one animal was dosed at 1000 mg/kg bw. A period of at least 48 hours was allowed between the dosing of each animal. Based on the results one additional animal was dosed at 2000 mg/kg bw. Based on the results of the range finding study, in the main study two additional animals were dosed at 2000 mg/kg bw. No mortality occurred. Red discoloration of the nose and ptosis were noted for one animal on Day 1. Fissures, scales, scabs and/or red discoloration were seen in the treated skin-area of the animals between Days 2 and 15. No abnormalities were found at macroscopic post mortem examination of the animals. The dermal LD50 value of Ethanol, 2,2’-[(3-methylphenyl)imino]bis- in Wistar rats was determined to exceed 2000 mg/kg body weight.

Justification for classification or non-classification

Based on the available data, the substance is classified as harmful if swallowed (Acute Tox. 4, H302) according to Regulation (EC) No. 1272/2008 (CLP). The substance does not need to be classified for acute dermal toxicity.