Reaction mass of 4-(2-methylbutan-2-yl)phenol and 2,4-bis(2-methylbutan-2-yl)phenol

Endpoint summary

Description of key information

Acute Toxicity: via oral route

Key value determined in a GLP accredited laboratory study using the Acute Toxic Class Method performed in accordance with OECD Guideline 423, EU Methos B1 Tris and US EPA Procedure OPPTS 870.1100. ssessment of acute oral toxicity with Phenol, 1,1-dimethylpropyl derivs. in the rat (Acute Toxic Class Method).

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:

15 September 2015 to 08 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Test type:

acute toxic class method

Limit test:

no

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. 8-12 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 affect 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 interfere with the study.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

Vehicle Rationale: Propylene glycol (Merck, Darmstadt, Germany) (specific gravity 1.036). The vehicle was selected based on trial preparations performed at WIL Research Europe and on test substance data supplied by the Sponsor. There was no information available regarding the solubility or stability in vehicle. Preparation: The preparations (w/w) were kept at room temperature protected from light and were dosed within 4 hours after adding the vehicle to the test substance. 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 substance. The concentration of the test substance in vehicle was varied to allow constant dosage volume in terms of mL/kg body weight. Prior to weighing the test substance was heated in a water bath with a maximum temperature of 80.1°C for a maximum of 3 hours and 15 minutes. In order to obtain homogeneity, the test substance (preparations) were heated in a water bath with a maximum temperature of 81.3 ºC for a maximum of 15 minutes. The test substance (formulations) were allowed to cool to a temperature of maximally 40°C prior to dosing.Study design The toxicity of the test substance 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:

Groups of 3 females.

Control animals:

no

Details on study design:

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 substance. 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. The time of death was recorded as precisely as possible. Body weights: Days 1 (pre-administration), 8 and 15 and at death (if found dead 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 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.

Statistics:

No statistical analysis was performed (The method used is not intended to allow the calculation of a precise LD50 value).

Key result

Sex:

female

Dose descriptor:

LD50 cut-off

Effect level:

500 mg/kg bw

Based on:

test mat.

Sex:

female

Dose descriptor:

approximate LD50

Effect level:

>= 300 - <= 2 000 mg/kg bw

Based on:

test mat.

Mortality:

At 2000 mg/kg, all animals died, one animal was found dead on Day 2 and two animals were found dead on Day 5.At 300 mg/kg, no mortality occurred.

Clinical signs:

other: At 2000 mg/kg, lethargy, flat posture, hunched posture, uncoordinated movements, slow breathing, shallow respiration, piloerection, watery discharge from left eye, pale appearance, lean appearance, hypothermia and/or ptosis were noted for the animals betw

Gross pathology:

At 2000 mg/kg, abnormalities of the stomach (irregular surface), gastrointestinal-tract (distended with gas, contents: gelatinous, yellowish) and/or thymus (reduced in size) were found in the animals that died during the study, at macroscopic post mortem examination.At 300 mg/kg, abnormalities of the stomach (irregular surface) were found in three animals at macroscopic examination.Autolysis was noted for two of the animals found dead.

Other findings:

No further information specified in the study report.

MORTALITY DATA

TEST DAY	1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
HOURS AFTER TREATMENT	0	2	4
FEMALES 2000 MG/KG FEMALES 300 MG/KG FEMALES 300 MG/KG	- - -	- - -	- - -	1 - -	- - -	- - -	2 - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -	- - -

 

CLINICAL SIGNS

TEST DAY	MAX GRADE	1	1	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15
HOURS AFTER TREATMENT		0	2	4
FEMALES 2000 MG/KG
ANIMAL 1 Behaviour -Lethargy Posture -Hunched posture Gait/Motility -Uncoordinated movements Skin/Fur -Piloerection Various -Ptosis	(3)   (1)   (3)   (1)   (3)	-   1   -   1   -	1   1   2   1   1	1   1   2   1   1
ANIMAL 2 Behaviour -Lethargy Posture -Hunched posture Gait/Motility -Uncoordinated movements Skin/Fur -Piloerection Various -Pale -Lean -Ptosis -Hypothermia	(3)   (1)   (3)   (1)   (3) (1) (3) (1)	-   1   -   1   - - - -	1   1   1   1   - - 1 -	1   1   1   1   - - 1 -	-   1   1   1   - - - -	-   1   1   1   - - - -	1   1   1   1   1 1 - 1
ANIMAL 3 Behaviour -Lethargy Posture -Flat posture -Hunched posture Gait/Motility -Uncoordinated movements Breathing -Slow breathing -Shallow respiration Skin/Fur -Piloerection Secretion/Excretion -Watery discharge from eye (Eye left) Various -Pale -Lean -Ptosis -Hypothermia	(3)   (1) (1)   (3)   (1) (3)   (1)   (3)   (3) (1) (3) (1)	-   - 1   -   - -   1   -   - - - -	2   1 -   -   1 1   1   1   - - 1 -	1   - -   -   1 1   1   1   - - 1 -	-   - 1   1   - -   1   -   - - - -	-   - 1   1   - -   1   -   - - - -	1   - 1   1   - -   1   -   1 1 - 1
FEMALES 300 MG/KG
ANIMAL 4 Behaviour -Lethargy Posture -Flat posture -Hunched posture Gait/Motility -Uncoordinated movement Breathing -Shallow respiration Skin/Fur -Piloerection	(3)   (1) (1)   (3)   (3)   (1)	-   - 1   -   -   -	1   - 1   1   1   1	1   1 1   1   1   1	-   - 1   -   -   -	-   - -   -   -   -	-   - -   -   -   -	-   - -   -   -   -	-   - -   -   -   -	-   - -   -   -   -	-   - -   -   -   -	-   - -   -   -   -	-   - -   -   -   -	-   - -   -   -   -	-   - -   -   -   -	-   - -   -   -   -	-   - -   -   -   -	-   - -   -   -   -
ANIMAL 5 Behaviour -Lethargy Posture -Flat posture -Hunched posture Gait/Motility -Uncoordinated movement Breathing -Shallow respiration Skin/Fur -Piloerection Various -Ptosis	(3)   (1) (1)   (3)   (3)   (1)   (3)	-   - 1   -   -   -   -	1   - 1   1   1   1   1	1   1 1   1   1   1   1	-   - -   -   -   -   -	-   - -   -   -   -   -	-   - -   -   -   -   -	-   - -   -   -   -   -	-   - -   -   -   -   -	-   - -   -   -   -   -	-   - -   -   -   -   -	-   - -   -   -   -   -	-   - -   -   -   -   -	-   - -   -   -   -   -	-   - -   -   -   -   -	-   - -   -   -   -   -	-   - -   -   -   -   -	-   - -   -   -   -   -
ANIMAL 6 Behaviour -Lethargy Posture -Flat posture -Hunched posture Gait/Motility -Uncoordinated movement Breathing -Shallow respiration Skin/Fur -Piloerection Secretion/Excretion -Diarrhoea Various -Ptosis	(3)   (1) (1)   (3)   (3)   (1)   (1)   (3)	-   - 1   -   -   -   -   -	1   - 1   1   1   1   1   1	1   1 1   1   1   1   1   1	-   - -   -   -   -   -   -	-   - -   -   -   -   -   -	-   - -   -   -   -   -   -	-   - -   -   -   -   -   -	-   - -   -   -   -   -   -	-   - -   -   -   -   -   -	-   - -   -   -   -   -   -	-   - -   -   -   -   -   -	-   - -   -   -   -   -   -	-   - -   -   -   -   -   -	-   - -   -   -   -   -   -	-   - -   -   -   -   -   -	-   - -   -   -   -   -   -	-   - -   -   -   -   -   -
FEMALES 300 MG/KG
ANIMAL 7 Behaviour -Lethargy Posture -Hunched posture Gait/Motility -Uncoordinated movements Skin/Fur -Piloerection	(3)   (1)   (3)   (1)	-   1   -   -	-   1   1   -	1   1   1   1	-   1   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -
ANIMAL 8 Posture -Hunched posture Gait/Motility -Uncoordinated movements Skin/Fur -Piloerection	(1)   (3)   (1)	1   -   -	1   1   -	1   1   1	1   -   -	-   -   -	-   -   -	-   -   -	-   -   -	-   -   -	-   -   -	-   -   -	-   -   -	-   -   -	-   -   -	-   -   -	-   -   -	-   -   -
ANIMAL 9 Behaviour -Lethargy Posture -Hunched posture Gait/Motility -Uncoordinated movements Skin/Fur -Piloerection	(3)   (1)   (3)   (1)	-   1   -   -	-   1   1   -	1   1   1   1	-   1   -   -	-   1   -   -	-   1   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -	-   -   -   -

- = Sign not observed

 

BODY WEIGHTS (GRAM)

SEX/DOSE LEVEL	ANIMAL	DAY 1	DAY 8	DAY 15
FEMALES 2000 MG/KG	1 2 3   MEAN ST. DEV. N	198* 201** 184**   194 9 3	- - -   - - 0	- - -   - - 0
FEMALES 300 MG/KG	4 5 6   MEAN ST. DEV. N	182 179 165   175 9 3	206 194 177   192 15 3	216 204 188   203 14 3
FEMALES 300 MG/KG	7 8 9   MEAN ST. DEV. N	152 146 165   154 10 3	174 168 193   178 13 3	197 186 206   196 10 3

* Animal found dead on Day 2. Body weight at death: 191 gram.

** Animals found dead on Day 5. Body weights at death: 159 and 150 gram, respectively.

 

MACROSCOPIC FINDINGS

ANIMAL	ORGAN	FINDING	DAY OF DEATH
FEMALES 2000 MG/KG
1	General observations   Stomach	GI-tract: contents: gelatinous GI-tract: contents: yellowish Forestomach: irregular surface	Spontaneous death Day 2 after treatment
2	General observations	Advanced autolysis GI-tract: contents: yellowish	Spontaneous death Day 5 after treatment
3	General observations   Stomach Thymus	GI-tract: [distended with gas] Advanced autolysis Forestomach: irregular surface Reduced in size	Spontaneous death Day 5 after treatment
FEMALES 300 MG/KG
4	Stomach	Forestomach: irregular surface	Scheduled necropsy Day 15 after treatment
5	Stomach	Forestomach: irregular surface	Scheduled necropsy Day 15 after treatment
6	Stomach	Forestomach: irregular surface	Scheduled necropsy Day 15 after treatment
FEMALES 300 MG/KG
7		No findings noted	Scheduled necropsy Day 15 after treatment
8		No findings noted	Scheduled necropsy Day 15 after treatment
9		No findings noted	Scheduled necropsy Day 15 after treatment

 

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The oral LD50 value of Phenol, 1,1-dimethylpropyl derivs. 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 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 (2011) (including all amendments), Phenol, 1,1-dimethylpropyl derivs. 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 substances and mixtures (including all amendments), Phenol, 1,1-dimethylpropyl derivs. should be classified as Category 4 and should be labeled as H302: Harmful if swallowed.

Executive summary:

Assessment of acute oral toxicity with Phenol, 1,1-dimethylpropyl derivs. in the rat (Acute Toxic Class Method).

 

The study was carried out based on the guidelines described in:

OECD No.423 (2001) "Acute Oral Toxicity, Acute Toxic Class Method"

Commission Regulation (EC) No 440/2008, B1 tris: "Acute Oral Toxicity, Acute Toxic Class Method"

EPA, OPPTS 870.1100 (2002), "Acute Oral Toxicity"

JMAFF Guidelines (2000), including the most recent revisions.

 

Initially, Phenol, 1,1-dimethylpropyl derivs. was administered by oral gavage to three female Wistar rats at 2000 mg/kg body weight. In a stepwise procedure two additional groups of three females were dosed at 300 mg/kg body weight. The animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed on the day of death or after terminal sacrifice (Day 15).

 

At 2000 mg/kg, all animals died, one animal was found dead on Day 2 and two animals were found dead on Day 5.

At 300 mg/kg, no mortality occurred.

 

At 2000 mg/kg, lethargy, flat posture, hunched posture, uncoordinated movements, slow breathing, shallow respiration, piloerection, watery discharge from left eye, pale appearance, lean appearance, hypothermia and/or ptosis were noted for the animals between Days 1 and 5.

At 300 mg/kg, lethargy, flat posture, hunched posture, uncoordinated movements, shallow respiration, piloerection, ptosis and/or diarrhoea were noted for the animals on Days 1 and/or 2. Additionally, hunched posture was noted for one animal on Days 3 and 4.

 

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.

 

At 2000 mg/kg, abnormalities of the stomach (irregular surface), gastrointestinal-tract (distended with gas, contents: gelatinous, yellowish) and/or thymus (reduced in size) were found in the animals that died during the study, at macroscopic post mortem examination.

At 300 mg/kg, abnormalities of the stomach (irregular surface) were found in three animals at macroscopic examination.

 

The oral LD50 value of Phenol, 1,1-dimethylpropyl derivs. 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 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 (2011) (including all amendments), Phenol, 1,1-dimethylpropyl derivs. 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 substances and mixtures (including all amendments), Phenol, 1,1-dimethylpropyl derivs. should be classified as Category 4 and should be labelled as H302: Harmful if swallowed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

500 mg/kg bw

Justification for classification or non-classification

Acute Toxicity: via oral route

According to the OECD 423 test guideline, the LD50 cut-off value was considered to 500 mg/kg body weight.

Based on the results:

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments), Phenol, 1,1-dimethylpropyl derivs. 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 substances and mixtures (including all amendments), Phenol, 1,1-dimethylpropyl derivs. should be classified as Category 4 and should be labeled as H302: Harmful if swallowed.