Registration Dossier

Administrative data

Description of key information

Acute Oral Toxicity: LD50 = >2,000 mg/kg; OECD 420; Anon. (2017)

Acute Inhalation Toxicity: LC50 = >5.67 mg/L; OECD 436; Anon. (2017)

Acute Dermal Toxicity: Waiver

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:
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.
Qualifier:
according to
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
fixed dose procedure
Limit test:
yes
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
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals 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
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
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.

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

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The endpoint is concluded based on a single key study with a Klimisch rating of 1. No effects were observed up to the limit dose of 2,000 mg/kg.

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 July - 12 October 2017
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.
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.52 (Acute Inhalation Toxicity - Acute Toxic Class Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes
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: Stable - confirmed by HPLC.
- Solubility and stability of the test substance in the solvent/vehicle: Test item was aerosolised as supplied. For analysis, the collected test item was dissolved in methanol, in which it was stable and soluble.
- 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: The test item was aerosolized using a metal concentric jet nebulizer (Envigo CRS Limited, UK) located at the top of the exposure chamber.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): The test item was aerosolized using a metal concentric jet nebulizer (Envigo CRS Limited, UK) located at the top of the exposure chamber. The particle size analysis of the atmosphere drawn from the animals’ breathing zone concluded that the mean mass median aerodynamic diameter was 2.28 µm.

OTHER SPECIFICS: n/a
Species:
rat
Strain:
other: RccHan™ : WIST
Sex:
male/female
Details on test animals 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 old
- Weight at study initiation: 200-350 g
- Fasting period before study: No
- Housing: The animals were housed in groups of up to three by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Diet (e.g. ad libitum): Ad libitum - 2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK
- Water (e.g. ad libitum): Ad libitum - mains drinking water
- Acclimation period: At least 5 days

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

IN-LIFE DATES: From: 06 July 2017 To: 12 October 2017
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
2.28 µm
Geometric standard deviation (GSD):
1.58
Remark on MMAD/GSD:
The results of the particle size distribution should be viewed with caution and are included only as a guide to the aerosol/vapor ratio of the generated atmosphere, as the volatility of a test item will be over-emphasized by the high air velocities and low pressures within a cascade impactor.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nose only exposure apparatus.
- Exposure chamber volume: The cylindrical exposure chamber had a volume of approximately 30 liters (dimensions: 28 cm diameter x 50 cm high).
- Method of holding animals in test chamber: During the exposure period, each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring.
- Source and rate of air: Compressed air was supplied by means of an oil free compressor at a rate of 40 L /minute
- Method of conditioning air: Air was passed through a water trap and respiratory quality filters before it was introduced to the nebulizer.
- System of generating particulates/aerosols: Metal concentric jet nebulizer (Envigo CRS Limited, UK) located at the top of the exposure chamber.
- Method of particle size determination: The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). This device consisted of six impactor stages (10.4, 7.7, 4.1, 1.3, 0.90 and 0.56 μm cut points) with stainless steel collection substrates and a backup glass fiber filter, housed in an aluminum sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump. The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference. The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 10.4, 7.7, 4.1, 1.3, 0.90 and 0.56 μm was calculated. The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (percentage) of aerosol less than 4 μm (considered to be the inhalable fraction) was determined. An impinger, set up as per atmosphere concentration determination was placed in series after the cascade impactor. Submission of these samples for chemical analysis allowed an estimate to be made of the amount of atmosphere present in the vapor phase. The sampling procedure involved 2 liters of test atmosphere being drawn through a glass impinger containing methanol (made up to 80mL). The samples were then submitted for chemical analysis.
- Treatment of exhaust air: The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system.
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every 30 minutes throughout the 4-Hour exposure period. Oxygen levels within the exposure chamber were measured by an electronic oxygen analyzer (Servomex (UK) Ltd, Crowborough, East Sussex) located in a port in the animals breathing zone during the 4 Hour exposure period.

TEST ATMOSPHERE
- Brief description of analytical method used: The test atmosphere was sampled nine times during the exposure period. The sampling procedure involved 2 liters of test atmosphere being drawn through a glass impinger containing methanol (made up to 80mL). The samples were then submitted for chemical analysis (HPLC) using an external standard technique.
- Samples taken from breathing zone: yes

VEHICLE
- Composition of vehicle (if applicable): n/a
- Concentration of test material in vehicle (if applicable): n/a
- Justification of choice of vehicle: n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: Tabulated
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): Tabulated

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: Highest concentration stipulated for aerosols in OECD 436.
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
4 h
Concentrations:
Mean measured atmospheric concentration was 5.67 mg/L
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Individual body weights were recorded on arrival, prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs
Statistics:
Not required
Preliminary study:
During the characterization phase of the study, a group of two rats (one male, one female) were exposed to an atmosphere of the test item at a mean achieved atmosphere concentration of 1.82 mg/L for four hours. During exposure both animals exhibited decreased respiratory rate and wet fur. On removal from the chamber both animals decreased respiratory rate, hunched posture, pilo-erection, red/brown staining around the snout and wet fur. One hour post-exposure, decreased respiratory rate was no longer apparent. Both animals recovered to appear normal on Day 1 post-exposure.
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.67 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Remarks on result:
not determinable due to absence of adverse toxic effects
Mortality:
No mortality was observed.
Clinical signs:
Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-Hour inhalation studies. Wet fur is commonly recorded both during and for a short period after exposure. These observations are considered to be associated with the restraint procedure and, in isolation, are not indicative of toxicity. In addition to the observations considered to be due to the restraint procedure, the following abnormalities were detected: During exposure, on removal from the chamber and 1 hour post-exposure all animals exhibited decreased respiratory rate. All animals appeared normal on Day 1 post-exposure.
Body weight:
With the exception of one female, all animals showed body weight loss on Day 1 post-exposure. Animals showed expected gains in body weight during the remainder of the recovery period with the exception of one female that showed no gain in body weight from Days 3 to 7 post-exposure.
Gross pathology:
With the exception of dark patches on the lungs of two females, no abnormalities were noted at necropsy.
Other findings:
Not examined

Table 2       Concentrations, exposure conditions and mortality per animals treated

 Nominal Concentration

(mg/L)

 Analytical Concentration

(mg/L)

 MMAD

(µm)

 GSD

 Mortality (# dead/total)

 Male

 Female

 Combined

 11.91

 5.67

 2.28

 1.58

 0/3

 0/3

 0/6

Table 3       Individual clinical observations - (day of exposure)

 Mean achieved atmospheric concentration

(mg/L)

 Animal number and sex

 Effects noted during exposure

 Effects noted on removal from chamber

 Effects noted 1 hour post exposure

 1 hour

 2 hours

 3 hours

5.67 

1 male 

WfRd 

WfRd

WfRd 

WfRdHP 

WfRdHP 

2 male

WfRd

WfRd 

WfRd 

WfRdHP

WfRdHP 

3 male

 WfRd

WfRd 

WfRd 

WfRdHP 

WfRdHP 

4 female 

 WfRd

WfRd 

WfRd 

WfRdHP 

WfRdHP 

5 female

 WfRd

WfRd 

WfRd 

WfRdHP 

WfRdHP 

6 female

 WfRd

WfRd 

WfRd

WfRdHP 

WfRdHP 

H: hunched posture

P: pilo-erection

Rd: decreased respiratory rate

Wf: wet fur

Clinical signs were not observed in any of individuals beyond the completion of the exposure period.

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 436 (2017) - In an acute inhalation toxicity study, groups of young adult rats (RccHan:WIST, 3 males and 3 females) were exposed by inhalation route to Reaction Mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene for 4 hours to nose only at a concentration of 5.67 mg/L (limit test).  Animals then were observed for 14 days.

In the absence of mortality during the observation period, the inhalation LC50 was estimated to be greater than 5.67 mg/L.

In addition, with the exception of dark patches on the lungs of 2 female rats, there were no treatment related clinical signs, necropsy findings or changes in bodyweight observed in any of the individuals at the end of the observation period.

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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The endpoint is concluded based on a single key study with a Klimisch rating of 1. No effects were observed up to the limit dose of 5.67 mg/L.

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.5.3, Column 2 of REACH, testing by the dermal route does not need to be conducted if:
- the substance does not meet the criteria for classification as acutely toxic or STOT SE by the oral route and
- no systemic effects have been observed in in vivo studies with dermal exposure (e.g. skin irritation, skin sensitisation) or, in the absence of an in vivo study by the oral route, no systemic effects after dermal exposure are predicted on the basis of non-testing approaches (e.g. read across, QSAR studies)
For this substance, no systemic effects were observed in the in vivo acute oral toxicity study (OECD 420), or in the LLNA study (OECD 429) where 100 % (w/w) solution was tested at the highest dose level.
Endpoint conclusion
Endpoint conclusion:
no study available
Quality of whole database:
The acute dermal toxicity study was waived in accordance with Annex VIII, Section 8.5.3, Column 2.

Additional information

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 the test item 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.

OECD 436 (2017) - In an acute inhalation toxicity study, groups of young adult rats (RccHan:WIST, 3 males and 3 females) were exposed by inhalation route to the test item for 4 hours to nose only at a concentration of 5.67 mg/L (limit test).  Animals then were observed for 14 days. In the absence of mortality during the observation period, the inhalation LC50 was estimated to be greater than 5.67 mg/L. In addition, with the exception of dark patches on the lungs of 2 female rats, there were no treatment related clinical signs, necropsy findings or changes in bodyweight observed in any of the individuals at the end of the observation period.

In accordance with Annex VIII, Section 8.5.3, Column 2 of REACH, testing by the dermal route does not need to be conducted if:

- the substance does not meet the criteria for classification as acutely toxic or STOT SE by the oral route and

- no systemic effects have been observed in in vivo studies with dermal exposure (e.g. skin irritation, skin sensitisation) or, in the absence of an in vivo study by the oral route, no systemic effects after dermal exposure are predicted on the basis of non-testing approaches (e.g. read across, QSAR studies)

For this substance, no systemic effects were observed in the in vivo acute oral toxicity study (OECD 420), or in the LLNA study (OECD 429) where 100 % (w/w) solution was tested at the highest dose level.

Justification for classification or non-classification

The substance does not meet the criteria for classification under GHS or Regulation (EC) No 1272/2008 (CLP).