6,8-Dioxabicyclo[3.2.1]octan-4-one, (1S,5R)-

Endpoint summary

Description of key information

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

In the key acute inhalation toxicity study, conducted according to OECD Test Guideline 436 and in compliance with GLP, the concluded LC50 value for Cyrene™ was greater than 5.16 mg/L, equivalent to >5160 mg/m³, following 4-hour nose only exposure of rat to Cyrene™ aerosol at limit concentration (Envigo, 2018a).

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

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

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Deviations:

yes

Remarks:

animals were fasted approximately 21 - 22 h prior treatment

Qualifier:

according to guideline

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 or test system 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:

other: No clinical signs were observed throughout the entire observation period.

Gross pathology:

No macroscopic findings were recorded at necropsy.

Interpretation of results:

GHS criteria not met

Conclusions:

In the acute oral toxicity study, conducted according to OECD Test Guideline 423 and in compliance with GLP, the LD50 value for female rats was determined to be greater than 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

Value:

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

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

Guideline:

EU Method B.52 (Acute Inhalation Toxicity - Acute Toxic Class Method)

Version / remarks:

2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

RccHan™: WIST strain

Sex:

male/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 to 12 weeks old
- Weight at study initiation: 200 g to 350 g
- Fasting period before study: not specified
- 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

Mass median aerodynamic diameter (MMAD):

ca. 3.1 µm

Remark on MMAD/GSD:

The mean MMAD was 3.10 µm with 60.4% with an inhalable fraction <4 µm.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: nose-only exposure chamber
- 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 aluminium 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 (limit concentration)
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 behavioural 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:

other: 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

Body weight:

Surviving animals showed body weight loss on Day 1 post-exposure and gains in body weight, in line with the historic growth rate, during the remainder of the recovery period.

Gross pathology:

The following macroscopic abnormalities were detected at necropsy of the animal found dead after 120 minutes exposure:
Lungs – Abnormally red, dark patches.
The following macroscopic abnormalities were detected at necropsy of animals surviving to the end of the observation period:
Lungs – Pale, abnormally red, dark patches, dark red patches
Kidneys – Pale.
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, conducted according to OECD Test Guideline 436 and in compliance with GLP, the concluded LC50 value was greater than 5.16 mg/L following 4-hour nose only exposure of rat to Cyrene™ aerosol at limit concentration.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

5 160 mg/m³ air

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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

In the study, 3 and 6 female Wistar rats were given single oral gavage dose of 300 or 2000 mg/kg bw/day in water, respectively. Thereafter, the animals were observed for clinical signs of toxicity daily for 14 days. At the end of the observation period the animals were subject to necropsy.

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 Test Guideline 436 and in compliance with GLP, the concluded LC50 value for Cyrene™ was greater than 5.16 mg/L (Envigo, 2018a).

In the study, 3 male and 3 female Wistar rats were subject to single nose-only inhalation exposure to the test substance aerosol at limit concentration of 5.16 mg/L for 4 hours. Thereafter, the animals were observed for clinical signs of toxicity daily for 14 days. At the end of the observation period the animals were subject to necropsy.

Following the inhalation exposure, 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 in the lungs as well as pale kidneys were noted at necropsy of animals surviving to the end of the observation period. The observed abnormalities were considered likely to be due to local toxicity.  

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.