Registration Dossier

Administrative data

Description of key information

In the key acute oral toxicity, conducted according to OECD 423 Test Guideline and in compliance with GLP, the reported LD50 value for the registered substance, Cyrene™, was greater than 2000 mg/kg bw/day (Harlan, 2014).

In the key acute inhalation toxicity study, conducted according to OECD TG 436 and GLP, the reported LC50 value for Cyrene™ was greater than 5.16 mg/L (Envigo, 2018).

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:
03 - 29 Apr 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
adopted 17 December 2001
Deviations:
yes
Remarks:
animals were fasted approximately 21 - 22 h prior treatment
Qualifier:
according to
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Deviations:
yes
Remarks:
animals were fasted approximately 21 - 22 h prior treatment
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Deviations:
yes
Remarks:
animals were fasted approximately 21 - 22 h prior treatment
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B. V., Horst, Netherlands
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 173.2 - 191.7 g (females)
- Fasting period before study: animals were fasted approximately 21 to 22 h prior treatment
- Housing: In groups of three in Makrolon type-4 cages with wire mesh tops and standard softwood bedding, including paper enrichment
- Diet: Pelleted Teklad Rat-Mouse Diet 2914C, ad libitum
- Water: Community tap water from Itingen, Switerzland, ad libitum, analysis was performed
- Acclimation period: Five days for group 1, 7 days for group 2 and 12 days for group 3 under laboratory conditions, after health examination
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- 15- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE- Concentration in vehicle: The test item was formulated in purified water at a concentration of 0.03 g/mL or 0.2 g/mL.
MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw
DOSAGE PREPARATION: The dose formulations were prepared shortly before each treatment in tared glass beakers and weighed on a suitable precision balance. The formulations were homogenized using a magnetic stirrer.
Doses:
300 and 2000 mg/kg bw
No. of animals per sex per dose:
3 (300 mg/kg bw), 6 (2000 mg/kg bw)
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: observation for clinical signs: ca. 30 min and 1, 2, 3 and 5 h after treatment on test day 1; once daily during test days 2 - 15; observation of body weight: on test day 1 (prior treatment), 8 and 15
Statistics:
no statistical analysis was performed
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
All animals survived the scheduled treatment.
Clinical signs:
No clinical signs were observed throughout the entire observation period.
Body weight:
No effects on body weight development were noted throughout the study.
Gross pathology:
No macroscopic findings were recorded at necropsy.
Interpretation of results:
GHS criteria not met
Conclusions:
In an acute oral toxicity study (OECD 423) the LD50 for rats was determined to be >2000 mg/kg bw. There were no mortalities, clinical signs of toxicity or adverse necropsy findings.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
2 000 mg/kg bw

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:
12 February 2018 to 29 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)
Version / remarks:
2009
Deviations:
yes
Remarks:
Slightly higher limit concentration (5.16 mg/L) was tested than the recommended 5 mg/L; lower relative humidity within the exposure chamber for Group 1; higher than 4 µm MMAD for Group 1. These deviations did not have an impact on validity of the study.
Qualifier:
according to
Guideline:
EU Method B.52 (Acute Inhalation Toxicity - Acute Toxic Class Method)
Version / remarks:
2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Wistar
Remarks:
RccHan™: WIST strain
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 to 12 weeks old
- Weight at study initiation: 200 g to 350 g
- Fasting period before study:
- Housing: Housed in groups of up to three by sex in solid floor polypropylene cages with stainless steel lids, furnished with softwood flakes
- Diet: food was available ad libitum, except during exposure
- Water: water was available ad libitum, except during exposure
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only exposure chamer
- 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 each 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. Only the nose of each animal was exposed to the test atmosphere.
- Source and rate of air: 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. The test atmospheres were generated to contain at least 19% oxygen.
- Method of conditioning air: The concentration within the exposure chamber was controlled by adjusting the rate of the infusion pump.
- System of generating particulates/aerosols: The test item was aerosolized using a metal concentric jet nebulizer located at the top of the exposure chamber. The nebulizer was connected to a glass syringe attached to an infusion pump, which provided a continuous supply of test item under pressure, and to a metered compressed air supply.
- Method of particle size determination: The particle size of the generated atmosphere inside the exposure chamber was determined three times during each 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.
- 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 chamber was maintained under negative pressure. The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter located in a vacant port in the animals’ breathing zone of the chamber and recorded every 30 minutes throughout the 4 Hour exposure period.

TEST ATMOSPHERE
- Brief description of analytical method used: The test atmosphere was sampled nine times during each exposure period. A weighed glass fiber filter was placed in a filter holder and temporarily sealed in a vacant port of the exposure chamber in the animals’ breathing zone. A known quantity of the exposure chamber atmosphere was drawn through the filter using a vacuum pump. The samples were then submitted for chemical analysis. The nominal chamber concentration was calculated by dividing the mass of test item disseminated into the chamber by the total volume of air that flowed through the chamber during the exposure.
- Samples taken from breathing zone: yes


TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: The particle size of the generated atmosphere inside the exposure chamber was determined three times during each exposure period using a Marple Personal Cascade Impactor.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 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.

Analytical verification of test atmosphere concentrations:
yes
Remarks:
The test atmosphere was sampled nine times. The nominal chamber concentration was calculated by dividing the mass of test item disseminated into the chamber by the total volume of air that flowed through the chamber during the exposure.
Duration of exposure:
4 h
Concentrations:
Sighting test: 2.0 mg/L
Main test: target concentration: 5.0 mg/L
Mean achieved concentration: 5.16 mg/L
No. of animals per sex per dose:
three males and three females
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 (limit test only) and at the end of the observation period.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs at 1 hour after termination of exposure and subsequently once daily for up to 14 days. All animals, including the one that died, were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.
Statistics:
Data evaluations included the relationship, if any, between the animals’ exposure to the test item and the incidence and severity of all abnormalities including behavioral and clinical observations, necropsy findings, body weight changes, mortality and any other toxicological effects.
Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) of the test item was made.
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.16 mg/L air
Based on:
test mat.
Exp. duration:
4 h
Mortality:
One male animal died at 120 minutes following exposure.
Clinical signs:
Wet fur is commonly recorded both during and for a short period after exposure. This observation is considered to be associated with the restraint procedure and, in isolation, not indicative of toxicity.
In addition to the observation considered to be due to the restraint procedure, the following abnormalities were detected: Decreased respiratory rate was observed in surviving animals during exposure, on removal from the chamber and 1 hour post exposure. There was an isolated instance of ataxia on removal from the chamber. No significant abnormalities were noted on Day 1 post-exposure.
Body weight:
Surviving animals showed body weight loss on Day 1 post-exposure and expected gains in body weight during the remainder of the recovery period.
Gross pathology:
Abnormally red with dark patches lungs were noted at necropsy of the animal found dead after 120 minutes exposure.
Pale, abnormally red, dark patches, dark red patches and pale kidneys were noted at necropsy of animals surviving tot the end of the observation period.
The observed abnormalities were considered likely to be due to local toxicity.
Other findings:
It is noted that the mean mass median aerodynamic diameter (MMAD) was higher than the range given in test guidelines (1-4 µm). This deviation is considered to be due to the physical characteristics of the test item.
Interpretation of results:
GHS criteria not met
Conclusions:
In the acute inhalation toxicity study, condicted according to OECD TG 436 and GLP, the reported LC50 value was greater than 5.16 mg/L.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
5 160 mg/m³

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In the key acute oral toxicity, conducted according to OECD 423 Test Guideline and in compliance with GLP, the reported LD50 value for the registered substance, Cyrene™, was greater than 2000 mg/kg bw/day (Harlan, 2014).

Following single oral administration of 300 or 2000 mg/kg bw/day to rats, no mortality occurred during the 14 -day study period. No clinical signs of toxicity, body weight gain changes or macroscopic changes were noted in any of the animals during the study period or at necropsy examination.

In the key acute inhalation toxicity study, conducted according to OECD TG 436 and GLP, the reported LC50 value for Cyrene™ was greater than 5.16 mg/L (Envigo, 2018).

Following single nose-only inhalation exposure of 5.16 mg/L to rats for 4 hours, one mortality was observed at 120 minutes post exposure during the 14-day study period. Wet fur was noted in all animals.  One animal was found dead after 120 minutes exposure, decreased respiratory rate was observed in surviving animals and there was an isolated instance of ataxia.  No significant abnormalities were noted on Day 1 post-exposure. Surviving animals showed body weight loss on Day 1 post-exposure and expected gains in body weight during the remainder of the recovery period. Abnormally red with dark patches lungs were noted at necropsy of the animal found dead after 120 minutes exposure. Pale, abnormally red, dark patches, dark red patches and pale kidneys were noted at necropsy of animals surviving tot the end of the observation period. The observed abnormalities were considered likely to be due to local toxicity.  

Testing for the acute dermal route is not considered to be necessary, in accordance with Column 2 of REACH Annex VIII:

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

- no systemic effects have been observed in in vivo studies with dermal exposure (skin irritation, skin sensitisation).

On this basis, it can be concluded that there is no short-term hazard for the dermal exposure route.

Justification for classification or non-classification

Based on the available data, no classification is required for acute toxicity for Cyrene™ according to Regulation (EC) No 1272/2008.