Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene

Endpoint summary

Description of key information

Acute Oral LD₅₀ (Rat) > 2000 mg/kg bw (OECD 423)

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:

2018-07-10 to 2018-07-25

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)

Version / remarks:

17 December 2001

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. (United Kingdom); Lot No. A170524D
- 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 (15-25oC, ≤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
- Treatment of test material prior to testing: 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.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Colorless 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, 8 weeks old
- Weight at study initiation: 175 – 197 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" (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 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6 – 24.7°C
- Humidity (%): 33 – 80%
- Air changes (per hr): 15 – 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 am to 6.00 pm

IN-LIFE DATES: From: 2018-07-05 To: 2018-07-25

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

Propylene glycol with 1% polysorbate 80

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 material. On the basis of the trial formulations with the test material, and considering the use of the vehicle in the ensuing repeated dose studies, the vehicle used was propylene glycol with 1% polysorbate 80.
- Lot/batch no. (if required): a) Propylene glycol (1,2-Propanediol), Batch no: K49089078; b) Polysorbate 80, Batch no: BCBV5152
- Purity: Not specified

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

DOSAGE PREPARATION: The test material 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: 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. The test material did not cause mortality in the confirmatory group, so no further testing was required according to OECD 423 and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.tris.

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. Body weight was measured on Days -1, 0 and 7 and Day 14.
- Necropsy of survivors performed: yes, All animals were subjected to a necropsy and a macroscopic examination.
- Other examinations performed: clinical signs (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.), body weight

Statistics:

The method used was not intended to allow the calculation of a precise LD50 value.
The test material 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:

The test material did not cause mortality at the dose level of 2000 mg/Kg bw.

Clinical signs:

other: No clinical signs of toxicity were observed susbsequent to oral exposure to the test material at 2000 mg/Kg bw.

Gross pathology:

Focal thickness of the non-glandular stomach mucosa was observed in 3 out of 6 animalsexposed to 2000 mg/Kg bw of the test material.

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

4174

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

4175

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

4176

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

2

4177

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

4178

Symptom Free

+

+

+

+

+

+

+

+

+

+

+

+

+

20/20

4179

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	4174	205	197	228	248	-8	31	20	43
	4175	201	186	225	236	-15	39	11	35
	4176	202	187	223	229	-15	36	6	27
2	4177	199	190	229	249	-9	39	20	50
	4178	186	175	212	207	-11	37	-5	21
	4179	194	185	200	228	-9	15	28	34
Mean		197.8	186.7	219.5	232.8	-11.2	32.8	13.3	35.0
Standard Deviation		6.9	7.2	11.3	15.5	3.1	9.2	11.8	10.5

 

Table 3. Necropsy Findings
Cage No.	Animal Number	Necropsy Date/ Necropsy Day	External Observations	Internal Observations	Organ/Tissue
1	4174	24 July 2018 Day 14	No external observations recorded	No internal observations recorded	Not Applicable
	4175	24 July 2018 Day 14	No external observations recorded	No internal observations recorded	Not Applicable
	4176	24 July 2018 Day 14	No external observations recorded	No internal observations recorded	Not Applicable
2	4177	25 July 2018 Day 14	No external observations recorded	Thickness, focal, non-glandular mucosa	Stomach
	4178	25 July 2018 Day 14	No external observations recorded	Thickness, focal, non-glandular mucosa	Stomach
	4179	25 July 2018 Day 14	No external observations recorded	Thickness, focal, non-glandular mucosa	Stomach

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

Under the conditions of this study, the acute oral LD50 value for the test material was determined to be >2000 mg/Kg bw in female Crl:WI rats.

According to the GHS criteria, the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene]) can be ranked as "Category 5" or “Unclassified” for acute
oral exposure.

Executive summary:

In a key study, the single-dose oral toxicity of the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene) 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 propylene glycol with 1% polysorbate 80 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 anddaily 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. As normal body weight changes were observed from Day 0 up to Day 7, and no other animal showed a similar trend between Day 7 up to Day 14, this change was considered incidental and minimal and not treatment-related.Focal thickness of the non-glandular stomach mucosa was observed in 3 out of 6 animals exposed to the test material.

 

Under the conditions of this study, the acute oral LD₅₀value for the test material was determined to be >2000 mg/Kg bw in female Crl:WI rats.

 

According to the GHS criteria, the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene]) can be ranked 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

Additional information

Acute Oral Toxicity

In a key OECD Guideline 423 study, the single-dose oral toxicity of the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene) was evaluated in Crl:WI rats according to the acute toxic class method.

 

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 propylene glycol with 1% polysorbate 80 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. As normal body weight changes were observed from Day 0 up to Day 7, and no other animal showed a similar trend between Day 7 up to Day 14, this change was considered incidental and minimal and not treatment-related.Focal thickness of the non-glandular stomach mucosa was observed in 3 out of 6 animals exposed to the test material.

 

Under the conditions of this study, the acute oral LD₅₀ value for the test material was determined to be >2000 mg/Kg bw in female Crl:WI rats.

 

According to the GHS criteria, the test material (Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene]) can be ranked as "Category 5" or “Unclassified” for acute oral exposure.

Justification for classification or non-classification

Reaction mass of dipentene and (Z)-3,7-dimethylocta-1,3,6,-triene 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.