Fatty acids, C18-unsatd., reaction products with acrylic acid and polyethylenepolyamines

Endpoint summary

Description of key information

Concluded to be a skin sensitiser; OECD guideline 406 (1992); Maulan M., 2010.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted in accordance with international guidelines in a GLP testing laboratory.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss GLP Authority

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was conducted prior to the LLNA method being the REACH Annex VII-endorsed in vivo method. For this substance, the GPMT method was considered the most appropriate.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

Animals: Albino Dunkin Harley guinea pigs HsdPoc: DH SPF
Rationale: Skin reactions in the guinea pig are classically used for determining the potential of test items to induce delayed contact hypersensitivity. No valid non-animal model (in-vitro) is available at present for the test of contact sensitization.
Breeder: Harlan Laboratories BV, Kreuzelweg 53, 5961 NM Horst, Nederlands
Age at Pretest Start / Beginning of Acclimatization Period: 4 – 6 weeks, with
Body weights: ranging from 317 – 338g (pre-test)
Body Weight at Beginning of Acclimatization Period: Test and control group: 297 – 350g (Main)
Identification: Uniquely identified by cage number and corresponding individual ear tag.
Randomization: Selected by hand at time of delivery. No computer generated randomization program.
Acclimatization: Under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study. A certificate of health was provided by the animal supplier at the animal delivery and included in the raw data.

Animals were maintained with 10-15 air changes per hour, a room temperature of 22 +/- degrees centigrade, a relative humidity of 30-70 percent and an automatic controlled light cycle of 12 hours light and 12 hours dark.

Animals were housed individually in Makrolon type-4 cages with standard softwood bedding and had ad libitum access to mains drinking water and food (Pelleted standard Provimi Kliba 3418 guinea pig breeding/maintenance diet supplied by Provimi Kliba, Kaiseraugst, Switzerland)

Route:

other: Intra-dermal and epidermal, occlusive

Vehicle:

water

Concentration / amount:

The pretest screened intradermal concentrations of 50, 25, 15% test item in water and epidermal concentrations of 100, 75, 50, 25 % test item in water.
For the main study, a 50% intradermal concentration, 100 % epidermal induction concentration and a 25 % epidermal challenge concentration in water were used.

Route:

other: Epidermal, occlusive

Vehicle:

water

Concentration / amount:

The pretest screened intradermal concentrations of 50, 25, 15% test item in water and epidermal concentrations of 100, 75, 50, 25 % test item in water.
For the main study, a 50% intradermal concentration, 100 % epidermal induction concentration and a 25 % epidermal challenge concentration in water were used.

No. of animals per dose:

15 (With 3 Pretest and 15 main test 10 test/5 control)

Details on study design:

PREPARATION OF DOSE FORMULATIONS
The test item and vehicle or auxiliary compound were placed into a glass beaker on a tared Mettler balance and a weight/weight dilution was prepared. Homogeneity of the test item preparation was ensured and maintained during treatment using a magnetic stirrer. The preparations were made immediately prior to each dosing.

Dilutions of the test material, PR-4758 were prepared by serial dilution in vehicle. Distilled water was selected as the vehicle based on preliminary solubility studies. A preparation of 75% in distilled water was soluble and passed through a needle of 0.9 x 40mm.

TEST ITEM ADMINISTRATION
Intradermal Induction
The 50% concentration of test item used for the intradermal induction exposure was well-tolerated systemically and caused moderate skin irritation during the pretest.

Epidermal Induction
A 100% concentration of test item used for the epidermal induction exposure was the highest concentration causing mild irritation during the pretest.

Epidermal Challenge
The 25% concentration of the test item used for the challenge application was non-irritant.

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.
No necropsy was performed.

TREATMENT
The animal's fur was shaved with a fine clipper blade or equivalent just prior to the exposure. Intradermal injections or closed patches were applied to the animals as follows:
0.1 mL/site for the intradermal administrations
0.2 to 0.3 mL on a filter paper for the epidermal administrations
The filter patch was covered by a strip of aluminium foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressing was left in place for 24 (epidermal pretest and epidermal challenge) or 48 hours (epidermal induction).
Identical patching method was used for the epidermal pretest, epidermal induction and epidermal challenge.

PRETEST
Based on the results, the test item concentration of 50% was selected for intradermal induction and 100 % for epidermal induction in the main study.
Based on the results obtained the concentration selected for the epidermal challenge in the main study was 25% test material

MAIN TEST
Induction
Intradermal injection / 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 just within the boundaries of a 4 x 6 cm area in the clipped region as follows:
Test Group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.
2) The test item at 50% in purified water.
3) The test item at 50% 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) purified water
3) 1:1 (w/w) mixture of purified water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

Epidermal Induction / Test Day 8
One week after the intradermal injections, 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 undiluted test item 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 water 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.

Challenge / 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 non-irritating concentration of 25%, applied to the left flank) and the vehicle only (water applied to the right flank) using the same method as for the epidermal application. The volume of test item preparation and of vehicle applied was approximately 0.2 mL. The dressings were left in place for 24 hours.

Determination of Skin Reactions - 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 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.

The scoring system was performed by visual assessment of erythema and oedema. They were assessed as follows:

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.
Grading of all animals was done by positioning each animal under true-light (FL-LPE OsramD16 FH 28W/840 EP).

Challenge controls:

The control group were also challenged with an epidermal 25 % test material in Distilled water application.

Positive control substance(s):

yes

Remarks:

Alpha-Hexylcinnamaldehyde

Positive control results:

70% of the test animals showed discrete/patchy erythema at the 24- or 48-hour reading after the second challenge treatment with ALPHA-HEXYLCINNAMALDEHYDE at 3% (w/v) in PEG 300. No skin effect was observed in the control group. No deaths occurred. No toxic signs were evident in the guinea pigs of the control or test group

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0% Test material, vehicle only (Distilled water)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No skin reactions observed

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0% Test material, vehicle only (Distilled water). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reactions observed.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0% Test material, vehicle only (Distilled water)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No skin reactions observed

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0% Test material, vehicle only (Distilled water). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: No skin reactions observed.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25% test material in distilled water

No. with + reactions:

1

Total no. in group:

5

Clinical observations:

Discrete patch erythema in 1 animal only

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25% test material in distilled water. No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: Discrete patch erythema in 1 animal only.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25 % Test material in distilled water

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

None observed

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25 % Test material in distilled water. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None observed.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25% test material in distilled water

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

Moderate/confluent erythema

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% test material in distilled water. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Moderate/confluent erythema.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25% test material in distilled water

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

Moderate/confluent erythema in 9/10 animals, discrete/patchy erythema 1/10 animals

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% test material in distilled water. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Moderate/confluent erythema in 9/10 animals, discrete/patchy erythema 1/10 animals.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0% test material, Distilled water vehicle only

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions observed

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0% test material, Distilled water vehicle only. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reactions observed.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0% test material, Distilled water vehicle only

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No skin reactions observed

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0% test material, Distilled water vehicle only. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reactions observed.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

3 % ALPHA-HEXYLCINNAMALDEHYDE

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

Skin reactions

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

3 % ALPHA-HEXYLCINNAMALDEHYDE

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

Skin reactions

Remarks on result:

positive indication of skin sensitisation

Pre-test skin reaction results

Test	Intradermal pretest
Animal	Sex	Concentration (%)	Reaction reading
			24-hr test sites	24-hr control sites	48-hr test sites	48-hr control sites
372	Male	A = 50 B = 25 C = 15	2 b 2 b 2 b	-	-	-
Test	Epidermal Pretest
373	Male	D = 100 E = 75 F = 50 G = 25	1 1 1 0	-	1 1 1 0	-
374	Male	G = 25 D = 100 E = 75 f = 75	0 1 1 1	-	0 1 1 1	-

Where b= blanching

On the basis of these results, test animals were intradermally induced with 50% test material, epidermally induced with 100% test material and challenged with 25% test material in distilled water.

Epidermal Induction skin reaction results

Animal	Sex	Concentration (%)	Reaction reading
			24-hr test sites	24-hr control sites	48-hr test sites	48-hr control sites
Test	Skin reactions after epidermal induction (Negative control - Vehicle only)
375	Male	100% water	1	0	0	0
376	Male		0	0	0	0
377	Male		0	0	0	0
378	Male		0	0	0	0
379	Male		0	0	0	0
Test	Skin reactions after epidermal induction (Test group - 100%)
380	Male	50% intradermal 100% epidermal induction	2 s	0	2 sc	0
381	Male		2 s	0	2 sc	0
382	Male		2 sc	0	2 sc	0
383	Male		2 sc	0	1 sc	0
384	Male		2 sco	0	1 sc	0
385	Male		2 sc	0	1 sc	0
386	Male		2 sc	0	2 scf	0
387	Male		2 s	0	1 s	0
388	Male		2 sc	0	2 sc	0
389	Male		2 sc	0	1 s	0

Where s = scaling, c = crusts, o = odema and f = fissures

Epidermal challenge skin reaction results

Animal	Sex	Concentration (%)	Reaction reading
			24-hr test sites	24-hr control sites	48-hr test sites	48-hr control sites
Test	Skin reactions in Challenge (Negative control - Vehicle only)
375	Male	100% water 25% Challenge	1	0	0	0
376	Male		0	0	0	0
377	Male		0	0	0	0
378	Male		0	0	0	0
379	Male		0	0	0	0
Test	Skin reactions in challenge (Test group - 100%)
380	Male	50% intradermal 100% epidermal induction 25% Challenge	2	0	2	0
381	Male		2	0	2	0
382	Male		2	0	2	0
383	Male		2	0	2	0
384	Male		2	0	2	0
385	Male		2	0	2	0
386	Male		2	0	1	0
387	Male		2	0	2	0
388	Male		2	0	2	0
389	Male		2	0	2	0

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the results of this study, the test item should be classified as a skin sensitiser.

Executive summary:

In order to assess the cutaneous allergenic potential of PR-4758, the Maximization-Test was performed in 15 (10 test and 5 control) male albino Dunkin Hartley guinea pigs, in accordance with OECD Guideline No. 406 and the Commission Regulation (EC) No 440/2008, B.6. The intradermal induction of sensitization in the test group was performed in the nuchal region with a 50% dilution of the test item in Water and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the

test item at 100% one week after the intradermal induction. The animals of the control group were intradermally induced with water and FCA/physiological saline and epidermally induced with water under occlusion.

Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 25% in water and water alone under occlusive dressing.

Cutaneous reactions were evaluated at 24 and 48 hours after removal of the dressing.

No deaths occurred. All 10 test animals showed skin reactions after the challenge treatment with PR-4758 at 25% in water. No skin effect were observed in the control group.

Based on the results of this study, the test item should be classified as a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The data used as a basis for this endpoint was the guinea pig maximisation test (Magnusson & Kligman). This study did not generate data necessary to derive a DNEL. Based on a semi-quantitative potency analysis in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R8, this substance can be considered a "moderate sensitiser". This potency assessment was conducted in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Part E.

Migrated from Short description of key information:

Concluded to be a skin sensitiser; OECD guideline 406 (1992); Maulan M., 2010.

Justification for selection of skin sensitisation endpoint:

One key study exists for this substance and is suitable for endpoint conclusion.  The study was conducted in accordance with OECD guideline 406 and in accordance with GLP.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

No information is available on potential to cause respiratory sensitisation. The material is however positive in a Magnusson & Kligman study commissioned for USA as such the material should be viewed as a skin sensitiser for classification purposes

Migrated from Short description of key information:

No information available

Justification for classification or non-classification

The study presented on the sensitising properties of the substance (Mallaun, 2010) indicate the substance is a sensitiser. There is no unequivocal evidence to sub-categorise sensitisation under Category 1B, therefore standard Category 1 classification applies.

In summary, the following skin sensitisation classification is applied to the substance:

Skin Sensitisation Category 1, H317: May cause an allergic skin reaction.