Hydrocarbon waxes (petroleum), oxidized, Me esters

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 May 2017 to 27 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

temperature higher than 25 °C on multiple dates with no impact on results or integrity of the study (see below)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

yes

Remarks:

temperature higher than 25 °C on multiple dates with no impact on results or integrity of the study (see below)

GLP compliance:

yes (incl. QA statement)

Type of study:

Buehler test

Justification for non-LLNA method:

Since the test item had been determined to be surface active, and OECD Guideline 429 notes that false positives may result from testing with some surfactant-type chemicals, expert opinion considered it more appropriate to investigate skin sensitisation using the Buehler assay.

Test material

Test material information

Constituent 1

Reference substance name:

Confidential

IUPAC Name:

Confidential

Test material form:

solid

Details on test material:

- Appearance / physical state: Dark brown paste
- Storage conditions: Room temperature in the dark

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

HOUSING
- Animals were housed in groups of maximum 3 in polycarbonate cages. The flooring of the cages was covered with dust-free wood shavings and the top fitted a stainless steel lid containing with a feeding device and drinking device of 500 mL.
- The cages were placed in an air-conditioned animal holding facility.
- Air change: at least 10 cycles per hour
- Temperature: 22 ° C ± 3 ° C
- Relative humidity from 30 % to 70 %
- Lighting: circadian cycle (12 hrs light and 12 hrs darkness)

FOOD AND DRINK
- The drinking water (tap water from public distribution system) and food (ENVIGO, 2040C) were supplied ad libitum.
- Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas - Eurofins (FRANCE).

PREPARATION OF ANIMALS
- Seventy albino guinea pigs of Dunkin-Hartley strain, supplied by ENVIGO (Kreuzelweg 53, 5961 NM HORST The Netherlands) and 3 or 4 weeks old at the beginning of the main test were used.
- Prior to the test, the animals were kept for a minimum acclimatisation period of 5 days, under housing and diet conditions identical to those of the test.
- Before the experimentation process, animals were identified individually by marking with picric acid and by means of a numbered ring on the edge of one ear.
- The animals were carefully shorn before each test item application:
(a) On the inter-scapular zone for the induction phase,
(b) On the dorso-lumbar zone for the challenge phase.
- At least 3 hours before the first reading (challenge phase) animals were shorn a second time in this dorso­lumbar zone.
- The animals were weighed at the beginning of the study, before the second induction and at the end of the study.

ANIMAL WELFARE
- The animals were provided with suitable environmental enrichment (Tunnel).
- The study was designed and was conducted to cause the minimum of suffering or distress to the animals, according to the guidelines and to our internal animal welfare's procedure. In particular, in case of suffering, the animal could be euthaniSed after decision of the Study Director.
- At the end of the test, the animals were euthanized with sodium pentobarbital (Dolethal).

Study design: in vivo (non-LLNA)

Induction

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

0.5 mL

Day(s)/duration:

Days 0, 7 and 14 for 6 hours

Adequacy of induction:

other: neat test item

Challenge

Challenge 1

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: liquid oaraffin

Concentration / amount:

75 % test item (0.5 mL)

Day(s)/duration:

Day 28 for 6 hours

Adequacy of challenge:

highest non-irritant concentration

Challenge 2

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

other: liquid paraffin

Concentration / amount:

75 % test item (0.5 mL)

Day(s)/duration:

Day 35 for 6 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Ten males and ten females

Details on study design:

PREPARATION OF TEST ITEM
- For the purpose of the study, the test item was prepared immediately prior to dosing in liquid paraffin for the topical applications (pretests). This vehicle was chosen as it produced the most suitable formulation at the required concentration.
- The preparation of the test item at 75% in liquid paraffin (w/w) was a brown thick solution and the preparation at 50% in liquid paraffin (w/w) was a brown solution.

PRELIMINARY STUDY – MAXIMUM NON-IRRITANT CONCENTRATION (MNIC) DETERMINATION
- This test was carried out with a reduced number of animals, for the purpose of determining the maximal non-irritating test item concentration to be used for the challenge phase. Furthermore, this test evaluates the irritant potential of the test item, and defines, if possible, a mild to moderate irritant concentration to be used for the topical induction phase.
- Two female and two male guinea pigs were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore adhesive tape from 3M and Blenderm from 3M) for a period of 6 hours at 4 different concentrations: 100%, and diluted at 75 %, 50 % and 25 % in liquid paraffin.
- The animals treated at the concentrations of 100 %, and diluted at 75 %, 50 % and 25 % received 0.5 mL of the corresponding preparation.
- A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 hours after removal of the occlusive dressings. The skin reaction was observed and recorded according to the grades described hereafter.

MAIN STUDY
- Group 1 (control): 5 male guinea pigs identified C0227 to C0231; 5 female guinea pigs identified C0262 to C0266.
- Group 2 (test item treated): 10 male guinea pigs identified C0232 to C0241; 10 female guinea pigs identified C0267 to C0276.
- Group 3 (control): 5 male guinea pigs identified C0242 to C0246; 5 female guinea pigs identified C0277 to C0281.
- Group 4 (HCA treated): 5 male guinea pigs identified C0247 to C0251; 5 female guinea pigs identified C0282 to C0286.

RECHALLENGE
- Group 1-bis (naïve control): 5 male guinea pigs identified C0252 to C0256; 5 female guinea pigs identified C0287 to C0291.
- Group 1-ter (naïve control): 5 male guinea pigs identified C0257 to C0261; 5 female guinea pigs identified C0292 to C0296.
- The naïve control animals (1-bis & 1-ter) were only used for the re-challenge phase, if necessary.
- Note: The results of the 3 last positive groups (Reference substance: a-Hexylcinnamaldehyde CAS No. 101-86-0 - Tests No.20-22) carried out as method sensibility, are presented in appendix 2.

INDUCTION PHASE
- After shearing of the scapular zone, the 3 local applications were performed on DO, D7 and D 14 for 6 hours under occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore adhesive tape from 3M and Blenderm (from 3M).
- The animals of the control group 1 received 0.5 mL of liquid paraffin and the animals of the treated group 2 received 0.5 mL of the test item at 100 % (undiluted).
- The animals of the control group 3 received 0.5 mL of liquid paraffin and the animals of the treated group 4 received 0.5 mL of alpha Hexylcinnamaldehyde (HCA) at 100 %.
- The animals of the control groups 1-bis & 1-ter received 0.5 mL of liquid paraffin.

REST PHASE
- The animals were left untreated for 13 days.

FIRST CHALLENGE (Day 28)
- The experimental procedure of this phase was identical for Group 1 (Control) and Group 2 (test item treated.
- On the previously shorn dorso-lumbar zone, an application was made on either side of the spine, under occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore adhesive tape from 3M and Blenderm from 3M), was performed for 6 hours: one area containing 0.5 mL of the test item diluted at 75 % (MNIC = maximal non-irritant concentration) on the left flank and one area containing 0.5 mL of liquid paraffin on the right flank.
- The experimental procedure of this phase was identical for Group 3 (Control) and Group 4 (HCA Treated).
- On the previously shorn dorso-lumbar zone, an application was made on either side of the spine, under occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore adhesive tape from 3M and Blenderm from 3M), was performed for 6 hours: one area containing 0.5 mL of HCA diluted at 50 % (MNIC = maximal non-irritant concentration) on the left flank and one area containing 0.5 mL of liquid paraffin on the right flank.
- The experimental procedure of this phase was identical for Group 1-bis (Naïve control) and Group 1-ter (Naïve control). On the previously shorn dorso-lumbar zone, an application was made on either side of the spine, under occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore adhesive tape from 3M and Blenderm from 3M), was performed for 6 hours: one area containing 0.5 mL of liquid paraffin on each flank area.

SECOND CHALLENGE (DAY 35)
- The experimental procedure of this phase was identical for Group Ibis (Control) and Group 2 (test item treated). On the previously shorn dorso-lumbar zone, an application was made on either side of the spine, under occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore adhesive tape from 3M and Blenderm from 3M), was performed for 6 hours: one area containing 0.5 mL of the test item diluted at 75 % (MNIC = maximal non-irritant concentration) on the left flank and one area containing 0.5 mL of liquid paraffin on the right flank.
- The experimental procedure of this phase was identical for Group 1 (Control) and Group 4 (HCA treated). On the previously shorn dorso-lumbar zone, an application was made on either side of the spine, under occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore adhesive tape from 3M and Blenderm from 3M), was performed for 6 hours: one area containing 0.5 mL of HCA diluted at 50% (MNIC = maximal non-irritant concentration) on the left flank and one area containing 0.5 mL of liquid paraffin on the right flank.
- The experimental procedure of this phase was for Group 1-ter (Naïve control), according to this approach: on the previously shorn dorso-lumbar zone, an application was made on either side of the spine, under occlusive dressing (25 mm x 50 mm non-woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore adhesive tape from 3M and Blenderm from 3M), was performed for 6 hours: one area containing 0.5 mL of liquid paraffin on each flank area.

MACROSCOPIC EXAMINATIONS AND EVALUATION OF CUTANEOUS REACTIONS
- A macroscopic evaluation of the cutaneous reactions (erythema and oedema) was conducted and all the local or systemic reactions were recorded for animals of Group 1 (Control) and Group 2 (Treated):
(i) Approximately 24 hours after removal of the occlusive dressing, the cutaneous reactions were observed and graded according to the scale, given below.
(ii) Approximately 24 hours later (i.e. 48 hours after removal of the occlusive dressing), a second observation was made.
- The grading scale used was 0 = no visible change; 1 = discrete or patchy erythema; 2 = moderate and confluent erythema; 3 = intense erythema and swelling.

INTERPRETATION OF RESULTS
- All animals with scores above or equal to 1 during the challenge phase, were considered positive.
- The percentage of animals that showed a sensitivity contact potential is calculated 24 and 48 hours after the removal of the occlusive dressings.
- A comparison of the intensity and persistence of reactions at the test item challenge sites in the test and control animals permits identification of sensitisation reactions. If the test item at the maximum non-irritant concentration produces reactions in test group animals at the 24 or 48 -hour readings, these reactions are attributed to skin sensitisation. This pre-supposes that no similar reactions are observed in the test material challenge sites of any of the control group animals. If irritation is observed in the control group animals, only reactions in the test group animals that exceed the most severe reaction seen in the control group animals are attributed to skin sensitisation. The results are expressed in terms of incidence and severity of responses.
- Incidence Score: The number of test group animals showing skin reactions greater than the most
severe reaction observed in the control group animals, expressed as a fraction of the total number of test group animals.
- Severity Score: The sum of the values assigned to the skin responses at the test item challenge sites of the test and control group animals, at each evaluation, divided by the number of animals in the relevant group.

INTERPRETATION OF RESULTS
- The experimental sensitising potential of the test item is established according to the attached scale.
- A response in at least 15% of animals is regarded as positive.

Positive control substance(s):

yes

Remarks:

alpha Hexylcinnamaldehyde

Results and discussion

Positive control results:

- In the treated group with positive control, a slight to well defined erythema was noted in 50 % (5/10), 40 % (4/10) and 20 % (2/10) of the.animals, 24, 48 and 72 hours after the re-challenge phase, on the area challenged with alpha Hexylcinnamaldehyde at 50 %. These reactions were significant of a skin sensitising response and the study was considered valid.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75 % test item

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: 1st challange (Day 28)

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75 % test item

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: 1st challenge (Day 28)

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75 % test item

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: 2nd challenge (Day 35)

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75 % test item

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

none

Remarks on result:

other: 2nd challenge (Day 35)

Results 5

Reading:

rechallenge

Hours after challenge:

72

Group:

negative control

Dose level:

Liquid paraffin

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 6

Reading:

rechallenge

Hours after challenge:

24

Group:

positive control

Dose level:

50 % hexylcinnamaldehyde

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

slight to well defined erythema

Remarks on result:

positive indication of skin sensitisation

Remarks:

study valid

Results 7

Reading:

rechallenge

Hours after challenge:

48

Group:

positive control

Dose level:

50 % hexylcinnamaldehyde

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

slight to well defined erythema

Remarks on result:

positive indication of skin sensitisation

Remarks:

study valid

Results 8

Reading:

rechallenge

Hours after challenge:

72

Group:

positive control

Dose level:

50 % hexylcinnamaldehyde

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

slight to well defined erythema

Remarks on result:

positive indication of skin sensitisation

Remarks:

study valid

Any other information on results incl. tables

Maximal Non Irritant Concentration (MNIC) DETERMINATION

- At 24 hours after removal of the patches, discrete erythema was observed in the animals (4/4) at the tested concentration of 100%. No cutaneous reaction was noted in the animals (4/4) at the tested concentrations of 75 %, 50 % and 25 % (see Table 1, attached).

- At 48 hours after removal of the patches, no cutaneous reaction was noted in the animals (4/4) whatever the tested concentrations of 75 %, 50 % and 25 % (see Table 1, attached).

- In view of these results the concentration selected was 100 % for the 3 inductions of the main study and 75 % for the challenge phase.

 

MAIN STUDY – INDUCTION PHASE (GROUP 2)

- Discrete erythema was observed in all animals (20/20) 24 hours after each induction (see Appendix 4, attached).

 

MAIN STUDY – INDUCTION PHASE (GROUP 1)

- No cutaneous reaction was noted during the induction phase (see Appendix 4, attached).

 

MAIN STUDY – INDUCTION PHASE (GROUP 4)

- Discrete erythema was observed in all animals (10/10) 24 hours after the first induction and the second induction (see Appendix 4, attached).

- Moderate erythema was observed in nine animals (9/10) and discrete erythema was observed in one animal (1/10) 24 hours after the third induction (see Appendix 4, attached).

 

MAIN STUDY – INDUCTION PHASE (GROUP 3)

- No cutaneous reaction was noted during the induction phase (see Appendix 4, attached).

 

MAIN STUDY – INDUCTION PHASE (GROUPS 1bis AND 1ter)

- No cutaneous reaction was noted during the induction phase (see Appendix 4, attached).

 

MAIN STUDY – CHALLENGE PHASES GROUPS 1, 2, 3 and 4

- Overall results of the 1st challenge (reading at 24, 48 and 72 hours) are given in Tables 2 and 6 (attached).

- Individual scores of macroscopic evaluations performed during the 1st challenge phase are given in Tables 4, 8 & 10 (attached).

- In the treated group with test item (treatment dose of 75 %), no macroscopic cutaneous reactions attributable to allergy were observed during the examination following the removal of the occlusive dressing.

- In the control group of test item (associated with the treatment dose of 75 %), no cutaneous intolerance reactions were observed during the examination following the removal of the occlusive dressing.

- No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin.

- In the treated group with positive control, no cutaneous reaction was noted on the area challenged with alpha Hexylcinnamaldehyde at 50 %.

- Due to the absence of reaction in the positive control group, a re-challenge was performed under the same experimental conditions after a rest phase of 7 days.

- Overall results of the 2nd challenge (reading at 24, 48 and 72 hours) are given in Tables 3 and 7 (attached). Individual scores of macroscopic evaluations performed during the 2nd challenge phase are given in Tables 5 & 9 (attached).

- In the treated group with test item (treatment dose of 75 %), no macroscopic cutaneous reactions attributable to allergy were observed during the examination following the removal of the occlusive dressing.

- In the control group of test item (associated with the treatment dose of 75 %), no cutaneous intolerance reactions were observed during the examination following the removal of the occlusive dressing.

- No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin.

 

WEIGHT EVALUATION

- The weight gain of control animals (Groups 1 & 3) and treated animals (Groups 2 & 4) is presented in Tables 11 to 15 (attached).

- No abnormalities and no differences in the body weight between the control and the treated group were observed.

 

MORTALITY

- No mortality occurred during the main test.

 

CLINICAL SIGNS

- No abnormal clinical signs related to the administration of the test item were observed.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was determined to be non-sensitising under the conditions of the study.

Executive summary:

GUIDELINE

The experimental protocol was established from the O.E.C.D. Test Guideline 406 dated July 17^th1992, Method B.6 of the Council Regulation No. 440/2008 and US EPA OPPTS 870.2600 (March 2003).

 

METHODS

The aim of the study was to evaluate the possible allergenic activity of the test item after topical administration in guinea pigs. According the results of the pre-tests, the induction phase (3 topical applications at 100 %) under occlusive dressing was conducted with the test item to 20 Guinea pigs and a 13-day rest phase. The challenge phase conducted under occlusive dressing for 6 hours, consisted of a single topical application of the test item diluted at 75% in liquid paraffin and of a negative control (liquid paraffin). At the request of sponsor, a positive control group treated with a reference allergen alpha Hexylcinnamaldehyde was selected.

 

RESULTS

In the treated group with test item (treatment dose of 75%), no macroscopic cutaneous reactions attributable to allergy were observed during the examination following the removal of the occlusive dressing. In the control group oftest item (associated with the treatment dose of 75%), no cutaneous intolerance reactions were observed during the examination following the removal of the occlusive dressing. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin. In the treated group with positive control, no cutaneous reaction was noted on the area challenged with alpha Hexylcinnamaldehyde at 50%.

 

Due to the absence of reaction in the positive control group, a rechallenge was performed under the same experimental conditions after a rest phase of 7 days. In the treated group with test item (treatment dose of 75%), no macroscopic cutaneous reactions attributable to allergy were observed during the examination following the removal of the occlusive dressing. In the control group of test item (associated with the treatment dose of 75 %), no cutaneous intolerance reactions were observed during the examination following the removal of the occlusive dressing. No cutaneous reaction was recorded in animals from the treated and control groups after the challenge phase, on the treated area with liquid paraffin. In the treated group with positive control, a slight to well defined erythema was noted in 50 % (5/10), 40 % ( 4/10) and 20 % (2/10) of the animals, 24, 48 and 72 hours after the rechallenge phase, on the area challenged with alpha Hexylcinnamaldehyde at 50%. These reactions were significant of a skin sensitising response and show the study to be valid.

 

CONCLUSION

The test item was determined to be non-sensitising under the conditions of the study.