Reaction mass of (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene and cis-1-methoxy-4-(2-methoxypropan-2-yl)-1-methylcyclohexane

Endpoint summary

Description of key information

A dermal sensititsation test was conducted with guinea pigs to determined the potential for the test item to produced sensitisation after repeated topical applications (Anon. 2005). The study was conducted in accordance with OECD 406 and in accordance with GLP.

Undiluted test item was topically applied to twenty healthy test guinea pigs, three times each week for a three-week induction period. Twenty eight days after the first induction dose, a challege dose of the test item at its highest non-irritating concnetration (HNIC, detremined in the preliminary irritation screen to be a 50% w/w miture in mineral oil) was applied to a naive site on each guinea pig. A naive control group (ten animals) was maintained under the same environmental conditions and treated with the test item at challenge only. Aproximately 24 and 48 hours after each induction and challege dose, the animals were scored for erythema.

For induction phae, very faint to faint erythma (0.5 - 1 score) was noted at the test sites in both control and positive control groups. An increase in irritation was evident at all sites as well as desquamation at most sites following the 3rd and 6th inductions.

For Challege phase, very faint erythema was observed for 5/20 test sites and irritation was persiten at one site through 48 hours. The erythema score of 0.5 was noted in test groups, test native control animals and historical control animals.

Based on these findings and on the evalution system used, the test item does not meet the criteria for positive contact sensitisation. The positive response noted in the historical positive control validation study with alpha-Hexylacinnamaldehyde technical (HCA) validate the test system used in the study.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 June - 13 July 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelnes and in accorance with GLP. All guideline criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

Due to technical error, animal #25820 (test group) was inadvertently dosed on a naive site on the right flank rather than on the left flank for the 1st, 2nd abd 3rd inductions an over all induction then dosed on the left flank for the challenge phase

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

The study was conducted prior to the LLNA method becoming the default approach for skin sensitisation under EU chemical control legislation. At the time of conducting this study, the Buehler method was considered the most appropriate test to assess skin sensitisation potential.

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A

OTHER SPECIFICS: N/A

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
Source: Received from Elm Hill Breeding Laboratories, Chelmsford, MA
Sex/Strain//number: Male and female guinea pigs/Harley albino (n= 34)
Age/weight at study initiation: Young adults 357 - 455 grams
Housing: the animals were group housed in suspended stainless steel caging with mesh floor
Diet: pelleted purina guinea pig chow #5025
Water: Filtered tap water ad libitum
Acclimation period: 12 or 14 days

ENVIRONMENTAL CONDITIONS
Temperature: 17-23°C
Humidity: 40 - 66%
Photoperiod: 12- hour light/dark cycle

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

0.4 ml

Day(s)/duration:

6 hours

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: Mineral oil

Concentration / amount:

0.4ml

Day(s)/duration:

6 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Total Number of Animals: 34
Number of Groups: 3
Preliminary Irritation Group: 4
Test Groups: 20
Naive Control Group: 10

Details on study design:

RANGE FINDING TESTS:
A group of animals (x4) was used to determine the highest non-irritating concentration (HNIC) of the test substance prior to dosing. The fur was removed by clipping the dorsal area and flanks of each guinea pig. This area was divided into four test sites on each animal. The test substance was applied neat (100 % w/w) and also diluted with mineral oil to yield w/w concentrations of 75 %, 50 % and 25 %. Each concentration was applied (0.4 mL) to a test site using an occlusive 25 mm Hiltop Chamber. The sites were wrappes with non-allergenic Durapore adhesive tape. After 6 hours of exposure, the chambers were removed and the test sites were cleansed of any residue substance. Approximately 24 hours after application, each site was evaluated for erythema.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1
- Exposure period: 6-hours
- Test groups: 1 (20 animals)
- Control group: 1 (10 animals)
- Site: Left flank
- Frequency of applications: Three times per week
- Duration: Three weeks
- Concentrations: 100 % (w/w)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 28
- Exposure period: 6-hours
- Test groups: 1 (20 animals)
- Control group: 1 (10 animals)
- Site: naive site on right flank
- Concentrations: 50 % (w/w)
- Evaluation (hr after challenge): 24 and 48 hours

Challenge controls:

10 guinea pigs from the same batch of animals were maintained under identical conditions and were treated with the HNIC of the test substance (50 % w/w) at challenge only.

Positive control substance(s):

yes

Remarks:

alpha-Hexylcinnamaldehyde (HCA). Testing completed on 04 March 2005.

Positive control results:

Induction Phase (HCA applied Undiluted): Very faint to faint (0.5 - 1) erythema was noted for all positive control test sites during the induction phase.
Challenge Phase Positive Control (75:25 HCA:Mineral Oil): 4/9 positive control animals exhibited signs of sensitisation response (faint erythema [1]) 24 and 48 hours after challenge. Very faint erythema (0.5) was noted for four other sites after challenge.
Challenge Phase Naive Control (75:25 HCA:Mineral Oil): Very faint erythema (0.5) was noted for three of five positive control naive test sites 24 hours after challenge. Irritation persisted at one of these sites through 48 hours.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 % (w/w)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Very faint erythema

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 % (w/w)

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

Very faint erythema

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 % (w/w)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Very faint erythema

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50 % (w/w)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None noted

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

75% w/w HCA in mineral oil

No. with + reactions:

4

Total no. in group:

9

Clinical observations:

no reaction (score = 0) in 1/9 to very faint erythema (score = 0.5) in 4/9 to faint erythema (score = 1.0) in 4/9

Remarks on result:

positive indication of skin sensitisation

Remarks:

One PC animal was excluded from evaluation as the patch was removed at 24 hours in technical error.

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

75% w/w HCA in mineral oil

No. with + reactions:

4

Total no. in group:

9

Clinical observations:

no reaction (score = 0) in 1/9 to very faint erythema (score = 0.5) in 4/9 to faint erythema (score = 1.0) in 4/9

Remarks on result:

positive indication of skin sensitisation

Remarks:

One PC animal was excluded from evaluation as the patch was removed at 24 hours in technical error.

Induction Phase - Test Animals (100 % w/w test item)

Very faint to faint erythema (0.5 -1) was noted at all test sites during the induction phase. An increase in irritation was evident at all sites after each successive application with desquamation present at most sites following the third and sixth inductions. Due to the severity of irritation, the dose sites were relocated to naive sited at various intervals throughout the induction phase.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance is not considered to be a skin sensitiser.

Executive summary:

A dermal sensitisation test was conducted with guinea pigs (OECD 406 - Buehler Method, 1992).  The undiluted test substance was topically applied to twenty healthy test guinea pigs, three times per week, for a three week induction period.  Twenty-eight days after the first induction dose, a challenge dose of the test item at its highest non-irritating concentration (50 % w/w) was applied to a naive site on each guinea pig.  A control group was treated with the test substance at challenge only.  Twenty four and 48 h after each induction and chellenge dose, the animals were scored for erythema.

Induction Phase (100 % test item): Very faint to faint erythema (0.5-1) was noted at all test sites during the induction phase.  Due to cumulative increase in irritation, the dose sites were relocated to other sites throughout the induction phase.

Challenge Phase (50 % test item):  Very faint erythema (0.5) was noted for five of twenty test sites after challenge.  Irritation persisted at one of these sites through 48 hours.   No positive responses evident.

Induction Phase (Control): Very faint erythema (0.5) was noted at six of the ten control sites 24 hours after challenge.  Irritation cleared from all affected sites by 48 hours. No positive responses evident.

The study met all relevant validation criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

A dermal sensitisation test was conducted with guinea pigs (OECD 406 - Buehler Method, 1992).  The undiluted test substance was topically applied to twenty healthy test guinea pigs, three times per week, for a three week induction period.  Twenty-eight days after the first induction dose, a challenge dose of the test item at its highest non-irritating concentration (50 % w/w) was applied to a naive site on each guinea pig.  A control group was treated with the test substance at challenge only.  Twenty four and 48 h after each induction and chellenge dose, the animals were scored for erythema.

Induction Phase (100 % test item): Very faint to faint erythema (0.5-1) was noted at all test sites during the induction phase.  Due to cumulative increase in irritation, the dose sites were relocated to other sites throughout the induction phase.

Challenge Phase (50 % test item):  Very faint erythema (0.5) was noted for five of twenty test sites after challenge.  Irritation persisted at one of these sites through 48 hours.  

Induction Phase (Control): Very faint erythema (0.5) was noted at six of the ten control sites 24 hours after challenge.  Irritation cleared from all affected sites by 48 hours.

The study met all relevant validation criteria.

The substance does not meet the GHS or CLP classification criteria for skin sensitisation.