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Description of key information

Based on the results of the read across GPMT, the test substance, 'di-C16-18 satd. and C18-24-unsatd. AAEMIM-MS' is considered to be non-sensitising to the skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose for cross-reference:
data waiving: supporting information
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From April 01, 2009 to June 16, 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
as layed down in Commission Regulation (EC) No 440/2008 of 30 May 2008
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
GPMT study was already available with the read across substance
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
Albino Dunkin Hartley Guinea Pig, CRL:(HA)BR, SPF
- Age at study initiation: 4-6 weeks
- Weight at study initiation: pretest groups: 332 - 349 g, test and control group: 330 - 366 g
- Housing: individually in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, 4132 Muttenz, Switzerland).
- Diet: ad libitum, pelleted standard Provimi Kliba 3418 guinea pig breeding / maintenance diet batch no. 82/08, Provimi Kliba AG, 4303 Kaiseraugst, Switzerland
- Water: ad libitum, tap water as for human consumption
- Acclimation period: animals of test and control group were acclimatised, no data on acclimation length


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): automatically controlled light cycle of 12 h light and 12 h dark

Route:
intradermal and epicutaneous
Vehicle:
polyethylene glycol
Concentration / amount:
Induction: 0.5 % (V/V) in intradermal, 10 % (V/V) epicutaneous challenge: 1 % (V/V)
Concentration selection based on a small scale dose range studies
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Concentration / amount:
Induction: 0.5 % (V/V) in intradermal, 10 % (V/V) epicutaneous challenge: 1 % (V/V)
Concentration selection based on a small scale dose range studies
No. of animals per dose:
treatment group: 10
control group: 5
Details on study design:
RANGE FINDING STUDIES
Intradermal injection:
For intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of three guinea pig. Six days later intradermal injections (0.1 mL/site) were made into the clipped flank . One animal received spatially divided injections of 25, 15, and 10 % , one of 5, 2.5, and 1 % and one of 0.5 and 0.1 %. Concentrations were formulated in polyethylene glycol 300 (PEG 300). Dermal reactions were assessed 24 hours later.

Dermal application:
Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. Six days later, four patches of filter paper (3 x 3 cm) were saturated with the test item at 100% (technically the highest possible concentration to be applied sufficiently), 75%, 50% and 25% in PEG 300 and applied to the clipped and shaved flanks of the same guinea pigs. The amount or volume of test item preparation applied was approximately 0.2 g at 100%, 75% and 50% or 0.2 mL at 25%. The concentration causing mild (to moderate) local skin reaction and the concentration being the highest tested non-irritating concentration could not be determined after the epidermal pretest described above. Therefore, a second pretest was performed with two additional naive guinea pigs, treated in the same way as those described previously, with the concentrations of 15%, 10%, 5% and 1% in PEG 300, using an application volume of approximately 0.2 mL.

MAIN STUDY
A. INDUCTION EXPOSURE
- First stage (test day 1)
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made symmetrically in two rows on either side of the sine just within the boundaries of a 4 x 6 cm area in the clipped region.
-- Test groups:
1. 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
2. The test item at 0.5% in PEG 300
3. The test item at 0.5% in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
- Control group:
1. 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
2. PEG 300
3. 1:1 (w/w) mixture of PEG 300 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline

- Second stage - One week after the intradermal injections (test day 8), the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair prior to the application. A 2 x 4 cm patch of filter paper was saturated with the test item at 10% in PEG 300 and placed over the injection sites of the test animals. The volume of test item preparation applied was approximately 0.3 mL. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressings were left in place
for 48 hours. The epidermal application procedure described ensured intensive contact of the test item.

The guinea pigs of the control group were treated as described above with PEG 300 only, applied at a volume of approximately 0.3 mL.

The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

B. CHALLENGE EXPOSURE (test day 22)
The test and control guinea pigs were challenged two weeks after the epidermal induction application and were treated in the same way.

Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested nonirritating concentration of 1% (applied to the left flank) and the vehicle only (PEG 300 applied to the right flank) using the same method as for the epidermal application. The volume of test item preparation and vehicle alone applied was approximately 0.2 mL. The dressings were left in place for 24 hours.

C. OBSERVATION and SCORING
The test item skin area of the animals used for the epidermal pretest and challenge was depilated approximately 21 hours after the patches have been removed, using an approved depilatory cream (VEET Cream, Reckitt & Colman AG, 4123 Allschwil, Switzerland). The depilation was performed to clean the stratum corneum from the possible staining produced by the test item and to facilitate the reading of the possible skin reaction. The depilatory cream was placed on the patch sites and surrounding areas, and left on for up to 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages.

The scoring system was performed by visual assessment of erythema and oedema.
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling

Any other gross lesions not covered by this scoring system were described.

The grading method used for the pretest, induction and challenge was identical. It was performed 24 ± 2 hours after removal of the patches for the intradermal and epidermal pretest and induction and for challenge and repeated 24 ± 2 hours later (48-hour grades) for the epidermal pretest,
epidermal induction and challenge.

OTHER OBSERVATIONS
- Viability / Mortality: Daily from delivery of the animals to the termination of the test.
- Clinical Signs / Grading of Skin Response: Daily from delivery of the animals to the termination of test. Skin responses were graded during the pretest, induction and challenge periods.
- Body Weights: At delivery/acclimatization start, at the end of the pretest, at test day 1 (day of treatment) and at the termination of the study.

POSITIVE CONTROL (RELIABILITY CHECK):
- The sensitivity and reliability of the experimental technique employed was assessed by use of ALPHA-HEXYLCINNAMALDEHYDE which is recommended by the OECD 406 Guidelines and is known to have moderate skin sensitisation properties in the guinea pig strain. The results from the most recent test run (Harlan Laboratories Study C16261, performed from 08-Oct-2008 to 14-Nov-2008) were included in the study report.

STATISTICAL ANALYSIS
Descriptive statistics (means and standard deviations) were calculated for body weights. No inferential statistics were used.
Positive control substance(s):
yes
Remarks:
Alpha-hexylcinnamaldehyde, periodically tested, most recent test run performed from 08-Oct-2008 to 14-Nov-2008
Positive control results:
All test animals showed skin reactions after the challenge treatment with Alpha-hexylcinnamaldehyde at 3% in PEG 300. No skin effect was observed in the animals treated with Alpha-hexylcinnamaldehyde at 1% in PEG 300. No skin effect was observed in the control group.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
1%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
discrete patchy erythema
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
other: Vehicle (PEG) control in initiated animals
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Vehicle 9PEG) control in initiated animals
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
3% in PEG 300
No. with + reactions:
5
Total no. in group:
5
Remarks on result:
positive indication of skin sensitisation

RESULTS

Range finding studies:

Intradermal injection: All tested concentrations caused skin reactions:

- 25 and 10 %: moderate and confluent erythema (score 2) with swelling

- 10, 5, 2.5 and 1 %: moderate and confluent erythema (score 2)

- 0.5 %: discrete or patchy erythema (score 1)

- 0.1 %: discrete or patchy erythema (score 1) with blanching

Dermal application: The test item at a concentration of 1 % caused no skin reactions. Concentration depended skin reactions after application of 5 to 100 %:

- 100 and 75 %: at 24 and 48 h scoring intense erythema and swelling (score 3), at 48 h with scaling and necrosis

- 50 %: at 24 and 48 h scoring moderate and confluent erythema (score 2), at 24 with swelling, at 48 h with scaling and necrosis

- 25 %: at 24 h scoring in both animals moderate and confluent erythema (score 2), at 48 h scoring one animals with discrete or patchy erythema (score 1) with scaling and one animals with moderate and confluent erythema (score 2) with scaling and necrosis

- 15, 10 and 5 %: at 24 and 48 h scoring discrete or patchy erythema (score 1), at 48 h with scaling

- 1 %: at 24 and 48 h scoring no visible changes

Main study:

- Induction:

-- intradermal: The expected and common findings were observed in the control and test group after the different applications using FCA intradermally. These findings consisted of erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation.

-- epicutaneous: No erythematous or oedematous reaction was observed in the animals treated with PEG 300 only. Discrete or patchy (score 1) to moderate and confluent (score 2) erythema was observed in all animals at the 24- and 48 -hour scorings after treatment with the test item at 10% in PEG 300; in detail at 24 h scoring 8/10 animals with discrete or patchy erythema (score 1), 2/10 animals with moderate and confluent erythema (score 2), at 48 h scoring 9/10 animals with discrete or patchy erythema (score 1), 1/10 animals with moderate and confluent erythema (score 2). Scaling was observed in 70% of the test animals at the 48-hour reading.

- Challenge

-- test group: Discrete or patchy erythema (score 1) was observed in 1/10 of the test animals at the 24-hour scoring after treated with the test item at 1% in PEG 300. No local skin reaction was observed at the 48-hour scoring.

No skin reactions were observed in the test animals when treated with PEG 300 only.

-- control group: No skin reactions were observed in the animals when treated with PEG 300 alone or when treated with the test item at 1% in PEG 300.

OTHER OBSERVATIONS

- Viability / Mortality: There were no deaths during the course of the study, hence no necropsies were performed.

- Clinical Signs: No signs of systemic toxicity were observed in the animals.

- Body Weights: The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Interpretation of results:
other: CLP criteria not met
Conclusions:
Based on the results of the read across study, a similar non-sensitising behaviour can be expected for the test substance.
Executive summary:

A study was conducted to determine the skin sensitisation potential of the read across substance, 'di-C16-18 and C18-unsatd. AAEMIM-MS' (active: 100%) using Guinea Pig Maximisation test (GPMT) method, according to OECD Guideline 406, in compliance with GLP.A pre-test was conducted to determine non-irritating concentrations to be used in the main study.The sensitivity of the test system was assessed by use of the positive control substance Alpha-exylcinnamaldehyde.In the main test, one treated group of ten maleDunkin Hartley guinea pigsreceived 0.5% of read across substance in a PEG 300 and in an emulsion of Freund’s Complete Adjuvant (FCA)/physiological saline (50/50, w/w), on Day 1 by intradermal injections in the nuchal region and48 h topical applications of 10% in PEG 300 on Day 8,under occlusive conditions. A control group of five male guinea pigs were intradermally induced with PEG 300 and FCA/physiological saline on Day 1 and epidermally induced with PEG 300 under occlusion on Day 8. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test substance at 1% in PEG 300 and PEG 300 alone under occlusive dressing. Cutaneous reactions were evaluated at 24 and 48 h after removal of the dressing. No toxic signs were evident in the guinea pigs of the control or test group. No deaths occurred. Ten percent of the test animals showed discrete or patchy erythema at the 24 h scoring after the challenge treatment with test substance at 1% (w/v) in PEG 300; at the 48 h scoring the 10 animals were free of skin reactions. None of the 5 control animals showed skin reactions at challenge. Under the study conditions, the read across substance was considered to be non-sensitising to the skin (Arcelin, 2009). Based on the results of the read across study, a similar non-sensitising behaviour can be expected for the test substance, 'di-C16-18-satd. and C18-24-unstad. AAEMIM-MS'.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

A study was conducted to determine the skin sensitisation potential of the read across substance, 'di-C16-18 and C18-unsatd. AAEMIM-MS' (active: 100%) using Guinea Pig Maximisation test (GPMT) method, according to OECD Guideline 406, in compliance with GLP.A pre-test was conducted to determine non-irritating concentrations to be used in the main study.The sensitivity of the test system was assessed by use of the positive control substance Alpha-exylcinnamaldehyde.In the main test, one treated group of ten maleDunkin Hartley guinea pigsreceived 0.5% of read across substance in a PEG 300 and in an emulsion of Freund’s Complete Adjuvant (FCA)/physiological saline (50/50, w/w), on Day 1 by intradermal injections in the nuchal region and48 h topical applications of 10% in PEG 300 on Day 8,under occlusive conditions. A control group of five male guinea pigs were intradermally induced with PEG 300 and FCA/physiological saline on Day 1 and epidermally induced with PEG 300 under occlusion on Day 8. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test substance at 1% in PEG 300 and PEG 300 alone under occlusive dressing. Cutaneous reactions were evaluated at 24 and 48 h after removal of the dressing. No toxic signs were evident in the guinea pigs of the control or test group. No deaths occurred. Ten percent of the test animals showed discrete or patchy erythema at the 24 h scoring after the challenge treatment with test substance at 1% (w/v) in PEG 300; at the 48 h scoring the 10 animals were free of skin reactions. None of the 5 control animals showed skin reactions at challenge. Under the study conditions, the read across substance was considered to be non-sensitising to the skin (Arcelin, 2009). Based on the results of the read across study, a similar non-sensitising behaviour can be expected for the test substance, 'di-C16-18-satd. and C18-24-unstad. AAEMIM-MS'.

Justification for classification or non-classification

Based on the available study results from read across GPMT, the test substance, ' di-C16-18-satd. and C18-24-unsatd. AAEMIM-MS', does not warrant classification for skin sensitisation, according to the EU CLP criteria (Regulation 1272/2008/EC).