rac-(1R,4S,4aR,8R,8aS)-9-(dichloromethylidene)-8-hydroxyoctahydro-1,4-methanonaphthalen-5(1H)-one

Endpoint summary

Description of key information

- Oral: LD50 > 2000 mg/kg bw, female, rat, OECD 425, Zelenák 2014

- Inhalation: LC50 > 5.05 mg/L, male/female, rat OECD 403, Nagy 2014

- Dermal: LD50 > 2000 mg/kg bw, male/female, OECD 402, Zelenák 2014

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:

11 Mar 2014 to 08 Apr 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

December 2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

up-and-down procedure

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

RccHan

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 160-179 g
- Fasting period before study: Overnight prior to treatment
- Housing: Individually in Type II polypropylene/polycarbonate cages. Rodents were housed with deep wood sawdust bedding to allow digging and other normal rodent activities Lignocel bedding for laboratory animals was available to animals during the study
- Diet: Autoclavable complete diet for rats and mice – breeding and maintenance ad libitum (except for pre-dose overnight fast).
- Water: Tap water ad libitum
- Acclimation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
11 Mar 2014 to 08 Apr 2014

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

VEHICLE
- Amount of vehicle: 10 mL/kg bw

DOSAGE PREPARATION
Homogeneity of the test item in the vehicle was maintained during administration using a magnetic stirrer.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5 females

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical observations after 30 mins, 1, 2, 3, 4 and 6 hours after dosing, then once daily thereafter for 14 days. Body weights recorded on days -1 and 0 (prior to dosing), 7 and 14.
- Necropsy of survivors performed: Yes. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. All gross pathological changes were recorded for each animal on the post mortem record sheets.

Statistics:

Data was evaluated using the Acute Oral Toxicity (OECD Test Guidelines 425) Statistical Programme (AOT 425 Stat Pgm).

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 mortality

Clinical signs:

other: Hunched back (2/5) and piloerection (1/5). All animals were symptom free from Day 2 until at the end of the observation period.

Gross pathology:

No treatment-related effects.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the acute oral median lethal dose LD50 of the test item was greater than 2000 mg/kg bw in RccHan:WIST female rats.

Executive summary:

In this GLP compliant, OECD 425 study, the acute toxicity of the test substance was evaluated in a limit test conducted with 5 female rats (RccHan:WIST). Animals were treated with a single oral (gavage) dose of the test substance at a dose level of 2000 mg/kg bw followed by a 14 day observation period. The animals were fasted overnight prior to treatment and food was returned 3 hours after dosing. All animals were observed individually after dosing at 30 minutes, 1, 2, 3, 4 and 6 hours post treatment and once each day for 14 days thereafter. Body weight was measured on Day -1 (prior to removal of food), Day 0 (prior to administration) and weekly thereafter. All animals were examined macroscopically at the end of the study.

There were no deaths and treatment at a dose level of 2000 mg/kg bw caused hunched back (2/5) and piloerection (1/5). All animals were symptom free from Day 2. There were no treatment related changes in the body weights. There was no evidence of the macroscopic observations in animals terminated on Day 14.

Under the conditions of this study, the acute oral median lethal dose LD50 of the test item was greater than 2000 mg/kg bw in RccHan:WIST female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Quality of whole database:

GLP compliant, OECD 425 study

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:

15 Jul 2014 to 29 Jul 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

Adopted: 7 September 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

traditional method

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

CRL

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 401-535 g (males); 246-312 g (females)
- Fasting period before study: No
- Housing: 5 per sex in Type III solid floor cages with stainless steel mesh lids. Rodents were housed with deep wood sawdust bedding to allow digging and other normal rodent activities. Lignocel Bedding for Laboratory Animals was available to animals during the study.
- Diet: Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance, ad libitum, except during exposure.
- Water: Tap water ad libitum, except during exposure.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2-26.5
- Humidity (%): 30-72
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
15 Jul 2014 to 29 Jul 2014

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

3.01 µm

Geometric standard deviation (GSD):

2.27

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Rodent Exposure System
- Exposure chamber volume: 3.85 L (inner plenum)
- Method of holding animals in test chamber: The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animal’s nares to enter the exposure port.
- Rate of air flow in (inner plenum): 15.0 L/min
- Rate of air flow out (inner plenum): 29.9 L/min
- System of generating particulates/aerosols: The test item was aerosolised using two Dust Feed Systems located at the top of the exposure chamber. Compressed air was supplied by means of an oil-free compressor and passed through a suitable filter system prior to introduction to the dust generator.
- Method of particle size determination: The particle size of the test atmosphere was determined three times during the exposure period using a 7-stage impactor of Mercer style.
- Temperature, humidity, oxygen concentration in air chamber: 23.6°C, humidity not recorded, oxygen concentration 20.6%.

TEST ATMOSPHERE
- Brief description of analytical method used: A known volume of test atmosphere was passed through weighed GF10 glass fibre filters. The difference in the pre- and post-sampling weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration.
The nominal concentration was calculated by dividing the mass of test material disseminated into the chamber by the total volume of air that went through the chamber during the same period.
- Samples taken from breathing zone: Yes

TEST ATMOSPHERE
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 3.01 µm / 2.27

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

Target concentration 5 mg/L. Actual avarage concentration 5.05 ± 0.12 mg/L.

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: Observed once per hour during exposure, as soon as possible following removal from restrains at the end of exposure, 1 hour after exposure and twice daily for 14 days thereafter. Body weights were recorded prior to treatment on the day of exposure (Day 0) and on Days 1, 3, 7 and 14.
- Necropsy of survivors performed: Yes. After examination of the external appearance, the thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. Any gross macroscopic changes were recorded. Special attention was given to the respiratory tract for macroscopic signs of irritancy or local toxicity.

Statistics:

Not applicable (limit test, no mortalities).

Preliminary study:

Sighting Exposure:
The mean achieved atmosphere concentration was 5.03 mg/L. The MMAD (Mean Mass Aerodynamic Diameter) was 3.01 μm ± 2.31 (GSD [Geometric Standard Deviation]). There was no mortality.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.05 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

There was no mortality.

Clinical signs:

other: Wet fur and/or fur staining were recorded in animals on the day of exposure and in some occasions on the day following exposure. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered n

Body weight:

Normal body weight gain was noted for all exposed animals during the observation period, with the exception of one female where slight body weight loss was observed during period Day 1-3.

Gross pathology:

No treatment-related findings.

Table 1: Summary of acute study test atmosphere characteristics

Achieved concentration	5.05 ± 0.12 mg/L (n=17)
Nominal concentration	9.37.mg/L
Particle size MMAD; GSD	3.01 µm; 2.27 (n=3)
Inhalable fraction (% <4 µm)	63.6
	Cumulative Mass (%)
Particles<0.55µm	2.95 (n=3)
Particles0.55-0.96µm	8.06 (n=3)
Particles0.96-1.55µm	15.70 (n=3)
Particles1.55-2.11µm	26.29 (n=3)
Particles2.11-3.56µm	62.28 (n=3)
Particles3.56–6.66µm	85.83 (n=3)
Particles6.66-10.55µm	93.84 (n=3)
Particles>10.55µm	100.00
Flow rate in (Inner plenum) (whole system)	15.0 L/min
Flow rate out (outer cylinder)	29.9 L/min
Theoretical chamber equilibrium time (T99)	1 minute
Actual equilibrium time allowed	11 minutes
Temperature	23.6°C (maximum/minimum 22.5/24.0°C)
Humidity	Not measured
Oxygen content	20.6% (maximum/minimum 20.3/20.8%)

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study, no deaths occurred in a group of 10 rats exposed to a mean achieved concentration of 5.05 mg/L for 4 hours. The acute inhalation median lethal concentration (LC50) in CRL: (WI) Wistar strain rats is, therefore, considered to be greater than 5.05 mg/L.

Executive summary:

In this GLP compliant, OECD 403 acute inhalation toxicity study, 5 male and 5 female CRL: (WI) Wistar strain rats, were exposed to a mean achieved concentration of 5.05 mg/L test substance for 4 hours using a nose-only exposure system, followed by a 14 day observation period. The day of exposure was designated Day 0. Aerosol concentrations were measured gravimetrically. The particle size distribution of the test aerosol was determined regularly during the exposure period. Clinical observations and body weights were recorded throughout the study and at the end of the scheduled period the animals were euthanised and subjected to a gross examination post mortem.

The mean achieved atmosphere concentration was 5.05 mg/L. The MMAD (Mean Mass Aerodynamic Diameter) was 3.01 μm ± 2.27 (GSD [Geometric Standard Deviation]). There was no mortality. Wet fur and/or fur staining were recorded in animals on the day of exposure and in some occasions on the day following exposure. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered not to be of toxicological relevance. Only slight to moderate laboured respiration was noted for the exposed animals on the day of exposure. No clinical signs were noted from Day 1 until the end of the observation period. Normal body weight gain was noted for all exposed animals during the observation period, with the exception of one female where slight body weight loss was observed during period Day 1-3. There were no treatment-related changes at gross examination post mortem.

Under the experimental conditions of this study, no deaths occurred in a group of 10 rats exposed to a mean achieved concentration of 5.05 mg/L for 4 hours. The acute inhalation median lethal concentration (LC50) in CRL: (WI) Wistar strain rats is, therefore, considered to be greater than 5.05 mg/L.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating conc.

Value:

0.005 mg/m³ air

Quality of whole database:

GLP compliant, OECD 403 study

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Mar 2014 to 27 Mar 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

Adopted: February 24, 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

RccHan (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Young adults
- Weight at study initiation: 218-251 g
- Fasting period before study: None
- Housing: Individually in Type II polypropylene/polycarbonate cages. Rodents were housed with deep wood sawdust bedding to allow digging and other normal rodent activities.
- Diet: Autoclavable complete diet for rats and mice – breeding and maintenance, ad libitum
- Water: Tap water ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
11 Mar 2014 to 27 Mar 2014

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Remarks:

(moistened with a small amount of water)

Details on dermal exposure:

TEST SITE
- Area of exposure: Back
- % coverage: Approximately 10 % area of the total body surface
- Type of wrap if used: Sterile gauze pads were placed on the skin of rats at the site of application. These gauze pads were kept in contact with the skin by a patch with adhesive hypoallergenic plaster. The entire trunk of the animal was then wrapped with semi occlusive plastic wrap.

REMOVAL OF TEST SUBSTANCE
- Washing: Yes, using body temperature water
- Time after start of exposure: 24 hours

TEST MATERIAL
- Amount applied (volume or weight with unit): 2000 mg/kg bw
- For solids, paste formed: Yes (wetted with a small amount of water)

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

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: Clinical observations / viability and mortality check at 1 and 5 hours after dosing and daily for 14 days thereafter. Body weight recorded prior to dosing on Day 0 and on Days 7 and 14.
- Necropsy of survivors performed: Yes. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. Any gross macroscopic findings were recorded.

Key result

Sex:

male/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 mortality occurred during the study.

Clinical signs:

other: There were no adverse clinical signs noted in any animals throughout the study. No treatment related skin irritation was observed in any animal throughout the study.

Gross pathology:

There was no evidence of the test item-related observations at a dose level of 2000 mg/kg bw at necropsy.

Interpretation of results:

GHS criteria not met

Conclusions:

The median lethal dose after a single dermal administration was found to be greater than 2000 mg/kg bw in male and female RccHan:WIST rat.

Executive summary:

In this GLP compliant, OECD 402 acute dermal absorption study, five male and five female rats (RccHan:WIST) were treated with a single, semi occlusive dermal application of the test substance at a dose of 2000 mg/kg body weight. The test item was wettened and applied as supplied. The application period was 24 hours, followed by a 14-day observation period. Clinical observations along with a check of viability and mortality were performed on all animals at 1 and 5 hours after dosing and daily for 14 days thereafter. Body weight was measured prior to dosing on Day 0 and on Days 7 and 14. Rats were euthanized and subjected to a gross macroscopic examination at the end of the 2-week observation period (Day 14).

No mortality occurred during the 14-day observation period after a 24-hour dermal exposure at 2000 mg/kg bw. No adverse clinical signs were observed after treatment with the test item or during the 14 day observation period and no effects were observed at the site of application. One female animal showed slight body weight loss during the second week of the observation period. There was no evidence of the test item-related observations at a dose level of 2000 mg/kg bw at necropsy.

The median lethal dose (acute dermal LD50) after a single dermal administration was found to be greater than 2000 mg/kg bw in male and female RccHan:WIST rat.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Quality of whole database:

GLP compliant, OECD 402 study

Additional information

Acute oral toxicity

In a GLP compliant, OECD 425 study, the acute toxicity of the test substance was evaluated in a limit test conducted with 5 female rats (RccHan:WIST) (Zelenák, 2014). Animals were treated with a single oral (gavage) dose of the test substance at a dose level of 2000 mg/kg bw followed by a 14 day observation period. The animals were fasted overnight prior to treatment and food was returned 3 hours after dosing. All animals were observed individually after dosing at 30 minutes, 1, 2, 3, 4 and 6 hours post treatment and once each day for 14 days thereafter. Body weight was measured on Day -1 (prior to removal of food), Day 0 (prior to administration) and weekly thereafter. All animals were examined macroscopically at the end of the study.

There were no deaths and treatment at a dose level of 2000 mg/kg bw caused hunched back (2/5) and piloerection (1/5). All animals were symptom free from Day 2. There were no treatment related changes in the body weights. There was no evidence of the macroscopic observations in animals terminated on Day 14.

Under the conditions of this study, the acute oral median lethal dose LD50 of the test item was greater than 2000 mg/kg bw in RccHan:WIST female rats.

Acute toxicity inhalation

In a GLP compliant, OECD 403 acute inhalation toxicity study, 5 male and 5 female CRL: (WI) Wistar strain rats, were exposed to a mean achieved concentration of 5.05 mg/L test substance for 4 hours using a nose-only exposure system, followed by a 14 day observation period (Nagy, 2014). The day of exposure was designated Day 0. Aerosol concentrations were measured gravimetrically. The particle size distribution of the test aerosol was determined regularly during the exposure period. Clinical observations and body weights were recorded throughout the study and at the end of the scheduled period the animals were euthanised and subjected to a gross examination post mortem.

The mean achieved atmosphere concentration was 5.05 mg/L. The MMAD (Mean Mass Aerodynamic Diameter) was 3.01 μm ± 2.27 (GSD [Geometric Standard Deviation]). There was no mortality. Wet fur and/or fur staining were recorded in animals on the day of exposure and in some occasions on the day following exposure. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered not to be of toxicological relevance. Only slight to moderate laboured respiration was noted for the exposed animals on the day of exposure. No clinical signs were noted from Day 1 until the end of the observation period. Normal body weight gain was noted for all exposed animals during the observation period, with the exception of one female where slight body weight loss was observed during period Day 1-3. There were no treatment-related changes at gross examination post mortem.

Under the experimental conditions of this study, no deaths occurred in a group of 10 rats exposed to a mean achieved concentration of 5.05 mg/L for 4 hours. The acute inhalation median lethal concentration (LC50) in CRL: (WI) Wistar strain rats is, therefore, considered to be greater than 5.05 mg/L.

Acute dermal toxicity

In a GLP compliant, OECD 402 acute dermal absorption study, five male and five female rats (RccHan:WIST) were treated with a single, semi occlusive dermal application of the test substance at a dose of 2000 mg/kg body weight (Zelenák, 2014). The test item was wettened and applied as supplied. The application period was 24 hours, followed by a 14-day observation period. Clinical observations along with a check of viability and mortality were performed on all animals at 1 and 5 hours after dosing and daily for 14 days thereafter. Body weight was measured prior to dosing on Day 0 and on Days 7 and 14. Rats were euthanized and subjected to a gross macroscopic examination at the end of the 2-week observation period (Day 14).

No mortality occurred during the 14-day observation period after a 24-hour dermal exposure at 2000 mg/kg bw. No adverse clinical signs were observed after treatment with the test item or during the 14 day observation period and no effects were observed at the site of application. One female animal showed slight body weight loss during the second week of the observation period. There was no evidence of the test item-related observations at a dose level of 2000 mg/kg bw at necropsy.

The median lethal dose (acute dermal LD50) after a single dermal administration was found to be greater than 2000 mg/kg bw in male and female RccHan:WIST rat.

Justification for classification or non-classification

Based on the available toxicity data, classification for acute toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.