Reaction mass of (E)-3-methylcyclotetradec-5-en-1-one and (Z)-3-methylcyclotetradec-5-en-1-one

Endpoint summary

Description of key information

The primary skin irritation potential of KARMALONE was investigated according to OECD test guideline no. 404. The test item did not induce significant or irreversible damage to the rabbit skin.

The primary eye irritation potential of KARMALONE was investigated according to OECD test guideline no. 405. The test item did not induce significant or irreversible damage to the rabbit eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 jan 2004 to 30 mar 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: KARMALONE
Description: Colourless to pale yellow liquid
Batch number: TOT0300497
Purity: 90.9%
Stability of test item: Stable under storage conditions
Expiry date: 28-NOV-2004
Storage conditions: At room temperature (range of 20 ± 3 °C), light protected.
Safety precautions: Routine hygienic procedures were used to ensure the health and satety of the personnel.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Source: Elevage scientifique des Dombes F-01400 Chatillon sur Chalaronne / France
Number of animals per test : 3 (animals of both sexes were used)
Age at treatment: 12 weeks (male and females)
Identification: By unique cage number and corresponding ear number
Acclimatization: Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.
Allocation: Male No. 53 and Females Nos 54 and 55
Room number: 106 / RCC Ltd, Fullinsdorf
Conditions:
Standard Laboratory Conditions
Air-conditioned with target ranges for room temperature 17-23 °C, relative humidity 30-70 % and approximately 10-15 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges may have occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark. Music was played during the daytime light period.
Accommodation: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (RCC Ltd, Fullinsdorf) and haysticks 4646 (batch no. 0403, Provimi Kliba AG) were provided for gnawing.
Diet: Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch no. 86/03) provided by Provimi Kliba AG, CH-4303 Kaiseraugst. Results of analysis for contami­ nants are archived at RCC Ltd, ltingen.
Water: Community tap water from Fullinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are ar­ chived at RCC Ltd, ltingen.

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

o.5 mL of Karmalone undiluted

Duration of treatment / exposure:

4 hours

Observation period:

Approximately 1, 24, 48 and 72 hours, as well as 7 and 10 days after the removal of the dressing, gauze patch and test item.

Number of animals:

3(one male and two females)

Details on study design:

Four days before treatment, the left flank was clipped with an electric clipper, exposing an area of approximately 100 cm2 (1O cm x 1O cm). The skin of the animals was examined one day before treatment, and regrown fur of all animals was clipped again.
Animals with overt signs of skin injury or marked irritation which may have interfered with the interpretation of the results were not used in the test.
On the day of treatment, 0.5 ml of KARMAlONE was placed on a surgical gauze patch (ca. 4 cm x 4 cm). This gauze patch was applied to the intact skin of the clipped area. The patch was covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and anchored with tape.
The duration of treatment was 4 hours. Then the dressing was removed and the skin was flushed with lukewarm tap water to clean the application site so that any reactions (erythema) were clearly visible at that time.

Observations:
Viability/Mortality: Daily from acclimatization of the animals to the termination of test.
Clinical signs: Daily from acclimatization of the animals to the termination of test.
Body weights: At start of acclimatization, on the day of application and at termination of observation.

Irritation scores: The skin reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992 approximately 1, 24, 48 and 72 hours, as well as 7 and 10 days after the removal of the dressing, gauze patch and test item.
To allow further examination of the test sites animal Nos. 53 and 55 were re-clipped on completion of the 24-hour examination as well as No. 53 again on completion of the 72-hour reading.

Treatment of results:
Data are summarized in tabular form, showing the irritation scores for erythema and oedema for each individual animal at all observation intervals after patch removal, any lesions, a description of the degree and nature of irritation, corrosion or reversibility, and any other toxic effects observed.
To evaluate the irritation of the test item the mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. An animal is positive when the mean score is 2 or greater. The test is positive for irritation when at least 2 animals are positive for the same endpoint (erythema/eschar or oedema).

Necropsy:
No necropsy was performed on the animals sacrificed at termination of observation.
All rabbits were sacrificed by an intravenous injection of Vetanarcol into the ear vein at a dose of at least 1 mUkg body weight (equivalent to 162 mg sodium pentobarbitone/kg body weight) and discarded.

Irritation parameter:

erythema score

Basis:

animal: 53

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

erythema score

Basis:

animal: 54

Time point:

24/48/72 h

Score:

0.67

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

erythema score

Basis:

animal: 55

Time point:

24/48/72 h

Score:

1.33

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

erythema score

Basis:

animal: 53, 54 and 55

Time point:

other: 1 hour

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

erythema score

Basis:

animal: 53, 54 and 55

Time point:

24 h

Score:

>= 1 - <= 2

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

erythema score

Basis:

animal: 53, 54 and 55

Time point:

48 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

erythema score

Basis:

animal: 53, 54 and 55

Time point:

72 h

Score:

>= 0 - <= 1

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

erythema score

Basis:

other: 53, 54 and 55

Time point:

7 d

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

erythema score

Basis:

animal: 53, 54 and 55

Time point:

10 d

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

edema score

Basis:

animal: 53

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

edema score

Basis:

animal: 54

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

edema score

Basis:

animal: 55

Time point:

24/48/72 h

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

edema score

Basis:

animal: 53, 54 and 55

Time point:

other: 1 hour

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

edema score

Basis:

animal: 53, 54 and 55

Time point:

24 h

Score:

1

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

edema score

Basis:

animal: 53, 54 and 55

Time point:

48 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

edema score

Basis:

animal: 53, 54 and 55

Time point:

72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

edema score

Basis:

animal: 53, 54 and 55

Time point:

7 d

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritation parameter:

edema score

Basis:

animal: 53, 54 and 55

Time point:

10 d

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 10 days

Irritant / corrosive response data:

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. The mean erythema/eschar score of the three animals was 0.67, 0.67 and 1.33, respectively and the mean oedema score was 0.33 for all three animals.
Well-defined erythema was observed in all animals at the 1-hour reading and very slight to well-defined erythema was noted in all animals 24 hours after treatment. Very slight erythema was present in all animals at the 48-hour examination and persisted in one animal up to 72 hours after treatment.
Very slight swelling (oedema) was present in all animals at the 1- and 24-hour reading. Scaling was observed in one animal 72 hours and 7 days after treatment.
No abnormal findings were observed on the treated skin of any animal 10 days after treatment, the end of the observation time.

Other effects:

No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
No staining produced by the test item of the treated skin was observed.
The body weights of all rabbits were considered to be within the normal range of variability.

Body weight in grams:

Body weight in grams
AnimalNo.	Sex	First Day of Acclimatization	Day of Treatment	Last Day ofObservation
53	male	2225	2476	2723
54	female	2127	2306	2718
55	female	2098	2385	2635

Interpretation of results:

GHS criteria not met

Conclusions:

Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 2001), KARMALONE is considered to be "not irritating" to rabbit skin.

Executive summary:

The primary skin irritation potential of KARMALONE was investigated according to OECD test guideline no. 404. The test item was applied by topical semi-occlusive application of 0.5 ml to the intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours, as well as 7 and 10 days after removal of the dressing.

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after patch removal) for each animal for erythema/eschar grades and for oedema grades, separately. The mean erythema/eschar score of the three animals was 0.67, 0.67 and 1.33, respectively and the mean oedema score was 0.33 for all three animals.

The application of KARMALONE to the skin resulted in slight, early-onset and transient signs of irritation such as erythema, oedema and scaling. These effects were reversible and were no longer evident 10 days after treatment, the end of the observation period for all animals. The test item caused no staining of the treated skin. No corrosive effects were noted on the treated skin of any animal at any of the measuring intervals and no other clinical signs of substance related effects were observed.

Thus, the test item did not induce significant or irreversible damage to the skin.

Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 2001), KARMALONE is considered to be "not irritating" to rabbit skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb 2004 to 1 Apr 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

conducted under GLP conditions

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Version / remarks:

1992

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: KARMALONE
Description: Colourless to pale yellow liquid
Batch number: TOT0300497
Purity: 90.9%
Stability of test item: Stable under storage conditions
Expiry date: 28-NOV-2004
Storage conditions: At room temperature (range of 20 ± 3 °C), light protected.
Safety precautions: Routine hygienic procedures were used to ensure the health and satety of the personnel.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Source: Elevage scientifique des Dombes F-01400 Chatillon sur Chalaronne / France
Number of animals per test : 3 (animals of both sexes were used)
Age at treatment: 11-12 weeks (male) and 12-13 weeks (females)
Identification: By unique cage number and corresponding ear number
Acclimatization: Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.
Allocation: Male No. 16 and Females Nos 17 and 18
Room number: 106 / RCC Ltd, Fullinsdorf
Conditions:
Standard Laboratory Conditions
Air-conditioned with target ranges for room temperature 17-23 °C, relative humidity 30-70 % and approximately 10-15 air changes per hour. Room temperature and humidity were monitored continuously and values outside of these ranges may have occasionally occurred, usually following room cleaning. These transient variations are considered not to have any influence on the study and, therefore, these data are not reported but are retained at RCC. The animals were provided with an automatically controlled light cycle of 12 hours light and 12 hours dark. Music was played during the daytime light period.
Accommodation: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls. Wood blocks (RCC Ltd, Fullinsdorf) and haysticks 4646 (batch no. 0403, Provimi Kliba AG) were provided for gnawing.
Diet: Pelleted standard Provimi Kliba 3418 rabbit maintenance diet ad libitum (batch no. 86/03) provided by Provimi Kliba AG, CH-4303 Kaiseraugst. Results of analysis for contami­ nants are archived at RCC Ltd, ltingen.
Water: Community tap water from Fullinsdorf, ad libitum. Results of bacteriological, chemical and contaminant analyses are ar­ chived at RCC Ltd, ltingen.

Vehicle:

unchanged (no vehicle)

Controls:

no

Amount / concentration applied:

0.1 mL of the test material undiluted

Duration of treatment / exposure:

On the day of treatment, 0.1 ml of KARMAlONE was placed in the conjunctiva! sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of test item. The right eye remained untreated and served as the reference control. The treated eyes were not rinsed after instillation.

Observation period (in vivo):

The ocular reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992 at approximately 1, 24, 48 and 72 hours after instillation.

Number of animals or in vitro replicates:

3

Details on study design:

Treatment: The eyes of the animals were examined one day prior to test item administration.
Animals with overt signs of ocular injury or irritation which may have interfered with the interpretation of the results were not used in the test.
On the day of treatment, 0.1 ml of KARMAlONE was placed in the conjunctiva! sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of test item. The right eye remained untreated and served as the reference control. The treated eyes were not rinsed after instillation.
As it was suspected that the test item might produce irritancy, a single animal (one female) was treated first. As neither a corrosive effect nor a severe irritant effect was observed after the 1- and 24-hour examinations, the test was completed using the two remaining animals.

Observations
Viability/Mortality: Daily from acclimatization of the animals to the termination of test.
Clinical signs: Daily from acclimatization of the animals to the termination of test.
Body weights: At start of acclimatization, on the day of application and at termination of observation.

Irritation scores
The ocular reaction was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992 at approximately 1, 24, 48 and 72 hours after instillation.
When present, corrosion and/or staining of conjunctivae, sclerae and cornea by the test item were recorded and reported.

Treatment of results:
Data are summarized in tabular form, showing the irritation scores of each following parameters: corneal opacity (including the area affected, where applicable), iridic effects, chemosis, conjunctiva! and scleral reddening for each individual animal at all observation intervals. In addition, any lesions including the degree and nature of irritation, corrosion or reversibility, and any other toxic effects are presented.
To evaluate the irritation potential of the test item mean values were calculated for each individual animal. Mean Values represent the mean of all available numerical scores (with the exception of the sclerae) recorded for the same parameter in each animal at 24, 48 and 72 hours.
For EU Classification of ocular irritants (Commission Directive 2001/59/EC), the criteria from the Official Journal of the European Communities (O.J. L 225/1) was employed (see page 23).

Necropsy: No necropsy was performed on the animals sacrificed at termination of observation.
All rabbits were sacrificed by an intravenous injection of Vetanarcol into the ear vein at a dose of at least 1 mUkg body weight (equivalent to 162 mg sodium pentobarbitone/kg body weight) and discarded.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

animal: 16

Time point:

24/48/72 h

Score:

0

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

conjunctivae score

Basis:

animal: 17 and 18

Time point:

24/48/72 h

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

fully reversible within: 48 hours

Irritant / corrosive response data:

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.00, 0.33 and
0.33 for reddening and 0.00 for chemosis in all three animals, respectively.
No abnormal findings were observed in the cornea or iris of any animal at any of the measurement intervals.
Moderate reddening of the conjunctivae, slight to moderate swelling, slight ocular discharge and moderate reddening of the sclerae were observed in all three animals at the 1-hour reading. The reddening of the conjunctivae faded to grade 1 (slightly reddened) in the two females at the 24-hour examination.
No abnormal findings were observed in the treated eye of any animal 48 hours after treatment.

Other effects:

No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.
No staining of the treated eyes produced by the test item was observed.
No corrosion of the cornea was observed at any of the reading times.
The body weights of all rabbits were considered to be within the normal range of variability.

Bodv weioht in orams
AnimalNo.	Sex	First Day of Acclimatization	Day ofTreatment	Last Day of Observation
16	male	2099	2258	2331
17	female	2040	2201	2263
18	female	2096	2265	2356

Interpretation of results:

GHS criteria not met

Conclusions:

Based upon the referred classification (Commission Directive 2001/59/EC of August 06, 2001), KARMALONE is considered to be "not irritating" to the rabbit eye.

Executive summary:

The primary eye irritation potential of KARMALONE was investigated according to OECD test guideline no. 405. The test item was applied by instillation of 0.1 ml into the left eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test iteminstillation.

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris, redness and chemosis of the conjunctivae, separately. The individual mean scores for corneal opacity and iris were 0.00 for all three animals. The individual mean scores for the conjunctivae were 0.00, 0.33  and

0.33 for reddening and 0.00 for chemosis in all three animals, respectively.

The instillation of KARMALONE into the eye resulted in slight to moderate, early-onset and transient ocular changes, such as reddening of the conjunctivae and sclerae, discharge and chemosis. These effects were reversible and were no longer evident 48 hours after treatment. No abnormal findings were observed in the cornea or iris of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. No staining of the treated eyes by the test item was observed and no other clinical signs of test item related effects wereobserved.

Thus, the test item did not induce significant or irreversible damage to the rabbit eye.

Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 06, 2001), KARMALONE is considered to be "not irritating" to the rabbiteye.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based upon the referred classification criteria (Commission Directive 2001/59/EC of August 2001) and

according to the EU CLP regulation (No 1272/2008 and its adaption 286/2011), KARMALONE is considered to be "not irritating" to rabbit skin.

Based upon the referred classification (Commission Directive 2001/59/EC of August 06, 2001) and according to the EU CLP regulation (No. 1272/2008 and its adaption 286/2011), KARMALONE is considered to be "not irritating" to the rabbit eye.