Indeno[4,5-d]-1,3-dioxin, 4,4a,5,6,7,8,9,9b-octahydro-7,7,8,9,9-pentamethyl-

Endpoint summary

Description of key information

Acute toxicity:
- Oral LD50: > 2500 mg/kg bw according to OECD TG 423
- Inhalation LD50 >  15000 mg/m3 (route-to-route extrapolation from acute oral toxicity study)
- Dermal LD50: > 2000 (OECD TG 402)

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:

Between 14 April 2003 and 06 May 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with OECD TG 423 and according to GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

female

Details on test animals or test system and environmental conditions:

Female Sprague-Dawley CD (Crl: CD (SD) IGS BR) strain rats were supplied by Charles River (UK) Ltd, Margate, Kent, UK. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the study the animals were eight to twelve weeks of age. The bodyweights fell within an interval of ± 20 % of the mean initial bodyweight of the first treated group.

The animals were housed in groups of three in suspended solid-floor polypropylene cages furnished with woodflakes. With the exception of an overnight fast immediately before dosing and for approximately three to four hours after dosing, free access to mains drinking water and food (Certified Rat and Mouse Diet (Code 5LF2) supplied by International Product Supplies Limited, Wellingborough, Northants, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 % respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

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.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe.

Doses:

2000 mg/kg

No. of animals per sex per dose:

6 female

Control animals:

no

Details on study design:

In the absence of data suggesting the test material was toxic, 2000 mg/kg was chosen as the starting dose.

Groups of fasted animals were treated as follows:

Dose Level Concentration Dose Volume Number of Rats
(mg/kg) (mg/mL) (ml/kg) (female)
2000 200 10 3
2000 200 10 3

All animals were dosed once only by gavage, using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to the fasted bodyweight at the time of dosing. Treatment of animals was sequential. Sufficient time was allowed between each group to confirm the survival of the previously dosed animals.

The animals were observed for deaths or overt signs of toxicity ½, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days.

Individual bodyweights were recorded prior to dosing and seven and fourteen days after treatment.

At the end of the observation period the animals were killed by cervical dislocation. All animals were subjected to gross pathological examination. This consisted of an external examination and opening of the abdominal and thoracic cavities for examination of major organs. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Statistics:

None recorded.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 500 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths.

Clinical signs:

other: There were no signs of systemic toxicity.

Gross pathology:

No abnormalities were noted at necropsy.

Other findings:

None

Evaluation of Data

Data evaluations included the relationship, if any, between the exposure of the animal to the test material and the incidence and severity of all abnormalities including behavioural and clinical observations, gross lesions, bodyweight changes, mortality and any other toxicological effects.

Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test material was made as shown in the schematic diagram in Appendix 1 (attachment 1).

The results were evaluated according to Commission Directive 2001/59/EC for classification and labelling of dangerous substances and preparations.

Interpretation of results:

not classified

Remarks:

Migrated information The acute oral median lethal dose (LD50) of the test material in the female Sprague-Dawley CD strain rat was estimated as being greater than 2500 mg/kg bodyweight. Criteria used for interpretation of results: other: EU labelling regulations Commission Directive 2001/59/EC.

Conclusions:

The acute toxcity of the test material via the dermal route was assessed accpording to OECD Guideline 423. The acute oral median lethal dose (LD50) of the test material in the female Sprague-Dawley CD strain rat was estimated from the flow chart in Appendix 1 (attachment 1) as being greater than 2500 mg/kg bodyweight. As such, the test material does not meet the criteria for classification according to EU labelling regulations Commission Directive 2001/59/EC.

Executive summary:

The acute oral toxicity of the test material was tested following a single oral administration in the Sprague-Dawley CD strain rat according to OECD TG 423 Acute Toxic Class Method. A group of three fasted females was treated with the test material at a dose level of 2000 mg/kg bodyweight. This was followed by a further group of three fasted females at the same dose level. The test material was administered orally as a solution in arachis oil BP. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy. There were no deaths. There were no signs of systemic toxicity. All animals showed expected gains in bodyweight over the study period. No abnormalities were noted at necropsy.The acute oral median lethal dose (LD₅₀) of the test material in the female Sprague-Dawley CD strain rat was greater than 2500 mg/kg bodyweight. The test material does not meet the criteria for classification according to EU labelling regulations Commission Directive 2001/59/EC.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Value:

mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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:

Between 28 May 2003 and 11 June 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with OECD 402 and with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Five male and five female Sprague-Dawley CD (Crl: CD (SD) IGS BR) strain rats were supplied by Charles River (UK) Ltd, Margate, Kent, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non-pregnant. After an acclimatisation period of at least five days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the study the animals weighed at least 200 g, and were eight to twelve weeks of age. The weight variation did not exceed ± 20 % of the mean weight for each sex.

The animals were housed in suspended solid-floor polypropylene cages furnished with woodflakes. The animals were housed individually during the 24-hour exposure period and in groups of five, by sex, for the remainder of the study. Free access to mains drinking water and food (Certified Rat and Mouse Diet (Code 5LF2) supplied by International Product Supplies Limited, Wellingborough, Northants, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 % respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

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.

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

The appropriate amount of test material, moistened with distilled water, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area).

Duration of exposure:

24 hours

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

5 male
5 female

Control animals:

not required

Details on study design:

On the day before treatment the back and flanks of each animal were clipped free of hair.

A group of five male and five female rats was treated with the test material at a dose level of 2000 mg/kg.

The appropriate amount of test material, moistened with distilled water, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area). A piece of surgical gauze was placed over the treatment area and semi-occluded with a piece of self-adhesive
bandage. The animals were caged individually for the 24-hour exposure period. Shortly after dosing the dressings were examined to ensure that they were securely in place.

After the 24-hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with distilled water to remove any residual test material. The animals were returned to group housing for the remainder of the study period.

The animals were observed for deaths or overt signs of toxicity ½, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days.
After removal of the dressings and subsequently once daily for fourteen days, the test sites were examined for evidence of primary irritation and scored according to the following scale from Draize JH (1977) "Dermal and Eye Toxicity Tests" In: Principles and Procedures for Evaluating the Toxicity ofH ousehold Substances, National Academy of Sciences, Washington DC p.31:

EVALUATION OF SKIN REACTIONS
Erythema and Eschar Formation Value

No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to slight eschar formation (injuries in depth) 4

Oedema Formation

No oedema 0
Very slight oedema (barely perceptible) 1
Slight oedema (edges of area well-defined by definite raising) 2
Moderate oedema (raised approximately 1 millimetre) 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) 4

Any other skin reactions, if present were also recorded.

Individual bodyweights were recorded prior to application of the test material on Day 0 and on Days 7 and 14.

At the end of the study the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.

Statistics:

No statistical analysis was performed.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: 95% confidence limits not reported.

Mortality:

There were no deaths.

Clinical signs:

other: There were no signs of systemic toxicity.

Gross pathology:

No abnormalities were noted at necropsy.

Other findings:

Dermal Reactions
There were no signs of dermal irritation.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The acute toxicity of the test material via the dermal route was assessed according to OECD Guideline 402. The acute dermal median lethal dose (LD50) of the test material in the Sprague-Dawley CD strain rat was found to be greater than 2000 mg/kg bodyweight.

Executive summary:

An acute dermal toxicity of the test material in the Sprague-Dawley CD strain rat using OECD TG 402 has been performed. A group of ten animals (five males and five females) was given a single, 24-hour, semi-occluded dermal application of test material to intact skin at a dose level of 2000 mg/kg bodyweight. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy. Mortality. There were no deaths. Clinical Observations. There were no signs of systemic toxicity. Dermal Irritation. There were no signs of dermal irritation. Bodyweight. All animals showed expected gains in bodyweight over the study period. Necropsy. No abnormalities were noted at necropsy.

The acute dermal median lethal dose (LD₅₀) of the test material In the Sprague-Dawley CD strain rat was found to be greater than 2000 mg/kg bodyweight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

The acute toxicity for oral, inhalation and dermal route has been derived.

The acute oral toxicity of the test material was tested following a single oral administration in the Sprague-Dawley CD strain rat according to OECD TG 423 Acute Toxic Class Method. A group of three fasted females was treated with the test material at a dose level of 2000 mg/kg bodyweight. This was followed by a further group of three fasted females at the same dose level. The test material was administered orally as a solution in arachis oil BP. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy. There were no deaths. There were no signs of systemic toxicity. All animals showed expected gains in bodyweight over the study period. No abnormalities were noted at necropsy.The acute oral median lethal dose (LD₅₀) of the test material in the female Sprague-Dawley CD strain rat was greater than 2500 mg/kg bodyweight.

An acute inhalation toxicity study is not needed for substances for which acute oral and dermal toxicity values are available. In addition, using route to route extrapolation the inhalation LC50 can be derived as follows: an oral LD50 of 2500 mg/kg bw can be roughly converted into 150000 mg/per person by multiplying it with a person’s weight: (60 kg x 2500). An inhalation volume for one person during 4 h (standard exposure time in the OECD TG for acute inhalation is 5m3 (assuming 10m3/8h for workers). This means that an LC50 concentration in 1 m3 and 4 hours exposure is 30000 mg/m3 (150000mg/5m3). Taking into account that during that the absorption via the inhalation route is twice the oral route the LC50, the LC50 for inhalation could be 15000 mg/m3. The maximum saturated vapour pressure for the substance is 37 mg/m3 (0.36 Pa (Vap Pr. Nebulone) x 250 (MW) / 8.3 (R, gas constant) x 293 (°K)). This means that Nebulone cannot reach a concentration higher than 37 mg/m3. Therefore an LC50 for inhalation cannot be reached and no classification and labelling is needed for the acute inhalation route.

An acute dermal toxicity of the test material in the Sprague-Dawley CD strain rat using OECD TG 402 has been performed.A group of ten animals (five males and five females) was given a single, 24-hour, semi-occluded dermal application of test material to intact skin at a dose level of 2000 mg/kg bodyweight. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy. There were no deaths, signs of systemic toxicity or signs of dermal irritation. All animals showed expected gains in bodyweight over the study period. No abnormalities were noted at necropsy.

The acute dermal median lethal dose (LD₅₀) of the test material In the Sprague-Dawley CD strain rat was found to be greater than 2000 mg/kg bodyweight.

Justification for selection of acute toxicity – oral endpoint
One acute oral toxicity study is available, which is performed according to OECD guidelines and under GLP conditions. This study is adequate for covering this endpoint.

Justification for selection of acute toxicity – inhalation endpoint
The acute inhalation toxicity is derived using route to route extrapolation from the acute toxicity results, using 100% absorption. This assessment is considered to be sufficient adequate for covering this endpoint.

Justification for selection of acute toxicity – dermal endpoint
One acute dermal toxicity study is available, which is performed according to OECD guidelines and under GLP conditions. This study is adequate for covering this endpoint.

Justification for classification or non-classification

According to the criteria outlined in Annex I of 67/548/EEC and Annex VI of 1272/2008/EC, Nebulone does not have to be classified as acutely toxic by the oral, dermal and inhalation route.