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Diss Factsheets

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 April 2016 to 28 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
acute toxic class method
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-(2-methylpropylidene)bis[4,6-xylenol]
EC Number:
251-394-8
EC Name:
2,2'-(2-methylpropylidene)bis[4,6-xylenol]
Cas Number:
33145-10-7
Molecular formula:
C20H26O2
IUPAC Name:
2-[1-(2-hydroxy-3,5-dimethylphenyl)-2-methylpropyl]-4,6-dimethylphenol
Test material form:
solid: particulate/powder
Details on test material:
Test Item: 207366/A
Identification: Lowinox®22IB46
Appearance: White to cream coloured powder
Batch: WB44L0016
Purity/Composition: 99.7%
Test item storage: At room temperature
Stable under storage conditions until: 18 November 2018 (retest date)
Chemical name (IUPAC): 2,2’-(2-methylpropylidene)bis[4,6-xylenol]
CAS Number: 33145-10-7
pH (1% in water, indicative range): 8.0 – 7.2

Test animals

Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals or test system and environmental conditions:
Species: Rat, Wistar strain Crl:WI (Han) (outbred, SPF-Quality). Recognized by international guidelines as the recommended test system (e.g. OECD, EC).
Source: Charles River Deutschland, Sulzfeld, Germany.
Number of animals: 9 Females (nulliparous and non-pregnant). Each dose group consisted of 3 animals.
Age and body weight: Young adult animals (approx. 8-9 weeks old) were selected. Body weight variation did not exceed +/- 20% of the sex mean.
Identification: Earmark and tail mark
Health inspection: At least prior to dosing. It was ensured that the animals were healthy and without any abnormality that might have affected the study integrity.
Animal Husbandry Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle the photoperiod was between 07:00 and 19:00 hrs daily. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation: Group housing of 3 animals per cage in labeled Makrolon cages (MIV type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
Acclimatization period was at least 5 days before start of treatment under laboratory conditions.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: Free access to tap water.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could be considered to interfere with the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
Test Item Preparation
Vehicle: Propylene glycol (Merck, Darmstadt, Germany) (specific gravity 1.036)
Rationale: The vehicle was selected based on trial preparations performed at WIL Research Europe and on test item data supplied by the Sponsor.
Preparation: The preparations (w/w) were kept at room and were dosed within 4 hours after adding the vehicle to the test item. Homogeneity was assessed by visual inspection of the solutions and the formulations were stirred during dosing, which ensures homogeneity sufficient for these kinds of studies. Adjustment was made for specific gravity of the vehicle. No correction was made for purity of the test item. The concentration of the test item in vehicle was varied to allow constant dosage volume in terms of mL/kg body weight.The toxicity of the test item was assessed by stepwise treatment of groups of 3 females. The first group was treated at a dose level of 2000 mg/kg. 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.
Doses:
2000 mg/kg (10 mL/kg) body weight.300 mg/kg (10 mL/kg) body weight.
No. of animals per sex per dose:
The toxicity of the test item was assessed by stepwise treatment of groups of 3 females.
Control animals:
not specified
Details on study design:
The toxicity of the test item was assessed by stepwise treatment of groups of 3 females.
The first group was treated at a dose level of 2000 mg/kg.
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.
Treatment Method: Oral gavage, using plastic feeding tubes. The test item preparations were stirred on a magnetic stirrer during dosing.
Fasting: Animals were deprived of food overnight prior to dosing and until 3-4 hours after administration of the test item. Water was available ad libitum.
Frequency: Single dosage on Day 1.
Dose level: (volume) 2000 mg/kg (10 mL/kg) body weight. 300 mg/kg (10 mL/kg) body weight.

Observations
Mortality/Viability: Twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).
Body weights: Days 1 (pre-administration), 8 and 15 and at death (if found dead or sacrificed after Day 1).
Clinical signs: At periodic intervals on the day of dosing (Day 1) and once daily thereafter, until Day 15. The signs were graded according to fixed scales and the time of onset, degree and duration were recorded: Maximum grade 4: grading slight (1) to very severe (4) Maximum grade 3: grading slight (1) to severe (3) Maximum grade 1: presence is scored (1).
Necropsy: The 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 recorded.

Evaluation: The oral LD50 value of the test item was ranked within the following ranges: 0-5, 5-50, 50-300 or 300-2000 mg/kg b.w. or as exceeding 2000 mg/kg b.w. The LD50 cut-off value was established based on OECD guideline 423. No statistical analysis was performed (The method used is not intended to allow the calculation of a precise LD50 value). The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (including all amendments) and Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and packaging of items and mixtures (including all amendments).
Statistics:
No statistical analysis was performed.

Results and discussion

Effect levels
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
500 mg/kg bw
Based on:
test mat.
Mortality:
At 2000 mg/kg, all animals were sacrificed for humane reasons on Day 2. At 300 mg/kg, no mortality occurred.
Clinical signs:
At 2000 mg/kg, lethargy, tremor, hunched posture, uncoordinated movements, slow breathing, shallow respiration, piloerection, watery discharge from eyes, lean appearance and/or hypersensitivity to touch were noted for the animals on Days 1 and/or 2.
At 300 mg/kg, lethargy, tremor, hunched posture, uncoordinated movements, rales and/or piloerection were noted for the animals between Days 1 and 5.
Body weight:
At 2000 mg/kg, the animals showed body weight loss during the first two days of the study.
At 300 mg/kg, the body weight gain over the study period was considered to be similar to that expected for normal untreated animals of the same age and strain.
Gross pathology:
No abnormalities were found at macroscopic post mortem examination of the animals.
Other findings:
No other findings were specified.

Applicant's summary and conclusion

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The oral LD50 value of Lowinox® 22IB46 in Wistar rats was established to be within the range of 300-2000 mg/kg body weight.
Executive summary:

The objective of this study was to assess the toxicity of the test item when administered in a single dose to female rats at one or more defined dosages. Furthermore, the results of the study allowed the test item to be ranked according to most classification systems, currently in use.

This study should provide a rational basis for risk assessment in man. The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item. The study complied with the following guidelines:

- Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects. No.423, "Acute Oral Toxicity - Acute Toxic Class Method", 2001.

 

- Commission Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B1 tris: "Acute Oral Toxicity, Acute Toxic Class Method". Official Journal of the European Union No. L142, May 2008, including the most recent amendments.

 

- United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.1100, Acute Oral Toxicity. Office of Prevention, Pesticides and Toxic Items (7101), EPA 712-C-02-190, 2002.

 

- Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000; including the most recent partial revisions.

 

Female rats administered with 2000 mg/kg were all sacrificed for humane reasons on Day 2. Clinical signs included:lethargy, tremor, hunched posture, uncoordinated movements, slow breathing, shallow respiration, piloerection, watery discharge from eyes, lean appearance and/or hypersensitivity to touch were noted for the animals on Days 1 and/or 2. At 2000 mg/kg, the animals showed body weight loss during the first two days of the study.

No mortality occurred in any of the female rats that were received a single dose of 300 mg/kg. There were however clinical signs including lethargy, tremor, hunched posture, uncoordinated movements, rales and/or piloerection which was noted for the animals between Days 1 and 5. The body weight of the rats administered 300mg/kg were comparable to animals of the same age and strain.

No abnormalities were found at macroscopic post mortem examination of the animals. 

The oral LD50 value of Lowinox® 22IB46 in Wistar rats was 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 500 mg/kg body weight. Based on these results:

-according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), Lowinox® 22IB46 should be classified as: harmful if swallowed (Category 4) for acute toxicity by the oral route;

-according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Lowinox® should be classified as Category 4 and should be labelled as H302: Harmful if swallowed.