2-tert-butyl-1,4-dimethoxybenzene

Endpoint summary

Description of key information

Acute oral LD₅₀ (Rat) >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:

2018-05-23 to 2018-06-07

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:

yes

Remarks:

Deviations have no presumed impact on the outcome or integrity of the study

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Emerald Kalama Chemical Ltd (UK); Batch no: A180118C
- Expiration date of the lot/batch: 2019-06-06
- Purity test date: 2018-02-07

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (15-25°C, ≤70 RH%), under inert gas, Protected from humidity (tight closed container)
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
was freshly formulated at a concentration of 200 mg/mL in the vehicle (PEG 400 (poly ethylene glycol)), in the Pharmacy of Citoxlab Hungary Ltd. on the day of administration. The formulation container was stirred continuously up to finishing the treatment.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear, pale yellow liquid

Species:

rat

Strain:

Wistar

Remarks:

CRL:(WI)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young healthy adult rats, 11 weeks old
- Weight at study initiation: 220 – 229 g
- Fasting period before study: On the night before treatment, the animals were fasted. The food but not water was withheld during an overnight period.
- Housing: 3 animals / cage (Type II polypropylene/polycarbonate)
- Diet (e.g. ad libitum): ssniff®SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany (Batch no.: 883 29966, expiry date: 31 October 2018), ad libitum
- Water (e.g. ad libitum): tap water from the municipal supply, as for human consumption from 500 ml bottles, ad libitum.
- Acclimation period: at least 27 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 40 -78%
- Air changes (per hr): 15 – 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): Lighting 12 hours daily, from 6.00 am to 6.00 pm

IN-LIFE DATES: From: 2018-04-26 To: 2018-06-07

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/Kg bw
- Justification for choice of vehicle: The selection of the vehicle was made during trial formulations with the test item. In order of preference, recommended vehicles were: distilled water, 0.5 or 1% methyl cellulose, PEG 400, corn oil, sunflower oil or DMSO (Dimethyl sulfoxide). On the basis of the trial formulations with the test item, the vehicle used was PEG 400 (poly ethylene glycol). Although no historical control data is available for the OECD 423 study at the test facility, PEG 400 is used routinely both in acute and repeated dose studies without any complications.
- Lot/batch no. (if required): A0378559

MAXIMUM DOSE VOLUME APPLIED: 10 mL/Kg bw

DOSAGE PREPARATION (if unusual): The test item was freshly formulated at a concentration of 200 mg/mL in the vehicle, in the Pharmacy of Citoxlab Hungary Ltd. on the day of administration. The formulation container was stirred continuously up to finishing the treatment.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: A limit of 2000 mg/kg bw dose was selected as the starting dose level which was most likely to produce mortality in some of the dosed animals. Initially, three females (assigned to Group 1) were treated at a dose level of 2000 mg/kg bw. The test item did not cause mortality in this group and a second group (Group 2) was treated at the same dose level. No mortality was observed in the confirmatory group, therefore no further testing was required according to OECD 423 and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.tris test guidelines.

Doses:

Single oral gavage dose of 2000 mg/Kg bw

No. of animals per sex per dose:

6

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Individual observations were performed on the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

The body weight was recorded on the day before treatment (Day -1), on the day of the treatment (Day 0), Day 7 and Day 14.

- Necropsy of survivors performed: yes
All animals were subjected to a necropsy and a macroscopic examination. All animals were exsanguinated after verification of narcosis following an injection of pentobarbital sodium (Euthanimal 40%; Lot No.: 1609291-03, Expiry Date: 31 October 2019, Produced by: Alfasan International B.V.). After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. All gross macroscopic changes were recorded for each animal.
- Other examinations performed: clinical signs, body weight

Statistics:

The method used was not intended to allow the calculation of a precise LD50 value.
The test item was ranked into categories of Globally Harmonized Classification System (GHS (rev. 7) 2017).
Clinical signs, body weight, body weight gain and gross macroscopic data were tabulated.

Preliminary study:

Initially, three females (assigned to Group 1) were treated at a dose level of 2000 mg/Kg body weight (bw). The test material did not cause mortality in this group therefore
a confirmatory group (Group 2) was treated at the same dose level.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality was observed at a dose level of 2000 mg/Kg bw.

Clinical signs:

other: Treatment with the test material at 2000 mg/Kg bw did not cause any clinical signs.

Gross pathology:

No macroscopic findings were observed in Crl:WI wistar female rats given a single dose of the test material at 2000 mg/Kg bw by oral gavage.

Table 1. Clinical Observations

Cage No.

Animal Number

Observations

Observation days

Frequency

0

1

2

3

2

5

6

7-14

30^’

1h

2h

3h

4h

6h

1

3720

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

3721

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

3722

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

2

3723

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

3724

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

3725

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

+ = present

' = minute

h = hour (s)

Frequency of observation = number of occurrence of observation / total number of observations

 

Table 2. Body Weight Data
Cage No.	Animal Number	Body weight (g) Days				Body Weight Gain (g)
		-1	0	7	14	-1-0	0-7	7-14	-1-14
1	3720	246	227	256	272	-19	29	16	26
	3721	245	229	253	255	-16	24	2	10
	3722	237	221	252	257	-16	31	5	20
2	3723	230	221	240	239	-9	19	-1	9
	3724	232	220	265	273	-12	45	8	41
	3725	230	220	262	262	-10	42	0	32
Mean		236.7	223.0	254.7	259.7	-13.7	31.7	5.0	23.0
Standard Deviation		7.3	3.9	8.8	12.6	3.9	10.1	6.3	12.6

 

Table 3. Necropsy Findings
Cage No.	Animal Number	Necropsy Date/ Necropsy Day	External Observations	Internal Observations	Organ/Tissue
1	3720	06 June 2018 Day 14	No external observations recorded	No internal observations recorded	Not Applicable
	3721	06 June 2018 Day 14	No external observations recorded	No internal observations recorded	Not Applicable
	3722	06 June 2018 Day 14	No external observations recorded	No internal observations recorded	Not Applicable
2	3723	07 June 2018 Day 14	No external observations recorded	No internal observations recorded	Not Applicable
	3724	07 June 2018 Day 14	No external observations recorded	No internal observations recorded	Not Applicable
	3725	07 June 2018 Day 14	No external observations recorded	No internal observations recorded	Not Applicable

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

Under the conditions of this study, the acute oral LD50 value of the test material (2-tert-butyl-1,4-dimethoxybenzene) was determined to be above 2000 mg/Kg bw in female CRL:(WI) rats.

According to the GHS criteria, 2-tert-butyl-1,4-dimethoxybenzene can be classified as "Category 5" or “Unclassified” for acute oral exposure.

Executive summary:

In a key study, the single-dose oral toxicity of the test material (2-tert-butyl-1,4-dimethoxybenzene) was evaluated in Crl:WI rats according to the acute toxic class method (OECD Guideline 423).

 

Initially, three females (assigned to Group 1) were treated at a dose level of 2000 mg/Kg bw. The test material did not cause mortality in this group, therefore a confirmatory group (Group 2) was treated at the same dose level. The test material did not cause mortality in the confirmatory group, so no further testing was required as per Guideline recommendations.

 

A single oral treatment was carried out by gavage for each animal after an overnight food withdrawal. Food was made available again 3 hours after the treatment. The test material was administered formulated in formulated in PEG 400 (poly ethylene glycol) at a concentration of 200 mg/mL at a dose volume of 10 mL/kg bw.

 

Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Body weight was measured on Days -1, 0 and 7 and Day 14. All animals were subjected to a necropsy and a macroscopic examination.

 

No mortality was observed through the study period and there was no evidence of clinical toxicity subsequent to treatment at 2000 mg/Kg bw. Normal body weight gain was observed in the observation period. However, one animal exhibited slight body weight loss between Day 7 and Day 14. This change was considered incidental and minimal and not treatment-related. No macroscopic findings were observed in Crl: WI wistar female rats.

 

Under the conditions of this study, the acute oral LD50 value of the test material (2-tert-butyl-1,4-dimethoxybenzene) was determined to be above 2000 mg/Kg bw in female CRL:(WI) rats.

 

According to the GHS criteria, the test material (2-tert-butyl-1,4-dimethoxybenzene) can be classified as "Category 5" or “Unclassified” for acute oral exposure.

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

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a key study (Citoxlab, 2019), the single-dose oral toxicity of the test material (2-tert-butyl-1,4-dimethoxybenzene) was evaluated in Crl:WI rats according to the acute toxic class method (OECD Guideline 423).

 

Initially, three females (assigned to Group 1) were treated at a dose level of 2000 mg/Kg bw. The test material did not cause mortality in this group, therefore a confirmatory group (Group 2) was treated at the same dose level. The test material did not cause mortality in the confirmatory group, so no further testing was required as per Guideline recommendations.

 

A single oral treatment was carried out by gavage for each animal after an overnight food withdrawal. Food was made available again 3 hours after the treatment. The test material was administered formulated in formulated in PEG 400 (poly ethylene glycol) at a concentration of 200 mg/mL at a dose volume of 10 mL/kg bw.

 

Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Body weight was measured on Days -1, 0 and 7 and Day 14. All animals were subjected to a necropsy and a macroscopic examination.

 

No mortality was observed through the study period and there was no evidence of clinical toxicity subsequent to treatment at 2000 mg/Kg bw. Normal body weight gain was observed in the observation period. However, one animal exhibited slight body weight loss between Day 7 and Day 14. This change was considered incidental and minimal and not treatment-related. No macroscopic findings were observed in Crl: WI wistar female rats.

 

Under the conditions of this study, the acute oral LD₅₀ value of the test material (2-tert-butyl-1,4-dimethoxybenzene) was determined to be above 2000 mg/Kg bw in female CRL:(WI) rats.

 

According to the GHS criteria, the test material (2-tert-butyl-1,4-dimethoxybenzene) can be classified as "Category 5" or “Unclassified” for acute oral exposure.

Justification for classification or non-classification

2-tert-butyl-1,4-dimethoxybenzene does not meet the criteria to be classified for acute oral toxicity under EU Regulation (EC) No 1272/2008 (CLP). However, according to the GHS criteria, based on an oral LD₅₀ of >2000 mg/Kg bw, the test material can be classified as "Category 5" or “Unclassified” for acute oral exposure.