Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
2-tert-butyl-1,4-dimethoxybenzene
EC Number:
244-216-5
EC Name:
2-tert-butyl-1,4-dimethoxybenzene
Cas Number:
21112-37-8
Molecular formula:
C12H18O2
IUPAC Name:
2-tert-butyl-1,4-dimethoxybenzene
Test material form:
liquid
Details on test material:
Name: 2-tert-butyl-1,4-dimethoxybenzene
Public name: Vetimoss
EC number: 244-216-5
CAS number: 21112-37-8
Batch/Lot Number: A180118C
Description: Clear, pale yellow liquid
Expiry Date: 06 June 2019
Purity: 99.78%
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

Test animals

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

Administration / exposure

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.

Results and discussion

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.
Effect levels
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:
Treatment with the test material at 2000 mg/Kg bw did not cause any clinical signs.
Body weight:
In the observation period, normal body weight gain was observed. However, one animal showed slight body weight loss between Day 7 and Day 14. This change was considered incidental and minimal and not ascribed to treatment.
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.