Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Novemeber - 14 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

(R)-p-mentha-1,8-diene

EC Number:

227-813-5

EC Name:

(R)-p-mentha-1,8-diene

Cas Number:

5989-27-5

Molecular formula:

C10H16

IUPAC Name:

(4R)-4-isopropenyl-1-methylcyclohexene

Constituent 2

Reference substance name:

(4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

Cas Number:

30199-25-8

Molecular formula:

C11H20O

IUPAC Name:

(4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene

Test material form:

liquid

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ca. 2 - 8 ºC in the dark and under nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: applied undiluted
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): applied as supplied

OTHER SPECIFICS: n/a

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: body weight variation did not exceed ±20% of the mean body weight at the start of treatment
- Fasting period before study: yes
- Housing: housed in groups of up to 4 individuals in suspended solid-floor polypropylene cages furnished with woodflakes.
- Diet (e.g. ad libitum): free access to food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK)
- Water (e.g. ad libitum): free access to mains drinking water
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 ºC
- Humidity (%): 30 - 70 %
- Air changes (per hr): minimum of 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 : 12

IN-LIFE DATES: From: 28 November 2016 To: 14 December 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

VEHICLE
- Concentration in vehicle: n/a
- Amount of vehicle (if gavage): n/a
- Justification for choice of vehicle: n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

MAXIMUM DOSE VOLUME APPLIED: 2.26 mL/kg

DOSAGE PREPARATION (if unusual): applied unchanged

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose: maximum guideline required concentration - 2000 mg/kg

Doses:

2000 mg/kg

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: 0.5, 1, 2 and 4 hours after dosing then daily thereafter.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs and body weight

Statistics:

not required

Results and discussion

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

No deaths reported

Clinical signs:

No signs of systemic toxicity were noted during the observation period.

Body weight:

All animals showed expected gains in body weight over the observation period.

Gross pathology:

No abnormalities were noted at necropsy.

Any other information on results incl. tables

Table 2       Number of animals dead (and with evident toxicity)

Dose (mg/kg bw)	Mortality (# dead / total)			Time range of deaths (hours)	Number with evident toxicity (# / total)
	Male	Female	Combined		Male	Female	Combined
2000	-	0 / 5	0 / 5	n/a	-	0 / 5	0 / 5

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item did not meet the criteria for classification according to Regulation (EC) No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures.

Executive summary:

OECD 420 (2017) - In an acute oral toxicity study, a group of fasted, 8-12 week old female Wistar rats were given a single oral dose of Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene (limonene) and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene (ether MT) at a single dose rate of 2000 mg/kg bw (limit test) and observed for 14 days.

In the absence of mortality during the observation period, the oral LD₅₀ was estimated to be greater than 2000 mg/kg bw.

In addition, there were no treatment related clinical signs, necropsy findings or changes in body weight observed in any of the individuals.

Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene (limonene) and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene (ether MT) did not meet the criteria for classification according to Regulation (EC) No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixture.