Fatty acids, tall-oil, polymers with glycidyl neodecanoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 03, 2015 to December 04, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Due to its fatty acid nature, the substance is expected to yield fasle positive results in LLNA testing.

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl (9Z)-octadec-9-enoate; 2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl (9Z,12Z)-octadeca-9,12-dienoate; 2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl octadecanoate

EC Number:

617-001-2

Cas Number:

80207-00-7

Molecular formula:

not applicable

IUPAC Name:

2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl (9Z)-octadec-9-enoate; 2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl (9Z,12Z)-octadeca-9,12-dienoate; 2-hydroxy-3-[(2,2,3,5-tetramethylhexanoyl)oxy]propyl octadecanoate

Test material form:

liquid

Remarks:

Brown

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: LAB-ÁLL Bt. Budapest, 1174 Hunyadi u. 7.
- Weight range at the beginning of the study: 242 - 348 g.
- Fasting period before study: No.
- Housing: macrolon cages, V., with 5 animals/cage (57x36x22 cm).
- Diet: Cunigra Rabbit Diet Mixture produced by Bonafarm-Bábolna Diet Ltd., 2942 Nagyigmánd, Hungary, ad libitum.
- Water: tap water, ad libitum containing 50 mg/100 mL ascorbic acid.
- Acclimatisation period: 7 d.

ENVIRONMENTAL CONDITIONS
- Temperature: 20±3 °C.
- Humidity: 30 - 70%.
- Photoperiod: 12 h daily from 6 a.m. to 6 p.m. (artificial light).

IN-LIFE DATES: November 03, 2015 to December 04, 2015.

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

other: sunflower oil

Concentration / amount:

Induction phase:
Concentration for intradermal injection: 5% *,
Concentration for topical application: 100% (undiluted),
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Challenge

Route:

epicutaneous, occlusive

Vehicle:

other: sunflower oil

Concentration / amount:

Challenge phase:
Concentration for topical application: 100%**,
** highest non-irritant concentration based on preliminary assays.

No. of animals per dose:

Main test: 10 control animals and 20 treated animals.

Details on study design:

- Dose range finding study:
Four animals each were used for the three phases of the dose range finding study, which included testing for selection of doses for intradermal induction, epicutaneous induction and challenge exposure. For the intra-dermal application 0.1 mL formulated test substance was injected at concentrations of 5, 10, 25 and 50% (w/v). The test substance formulations were injected in duplicate, so each animal received two injections. At concentration of 50% moderate erythema was observed in both animals one hour after the patch removal. At concentrations of 25 and 10% well defined erythema was observable in the animals one hour after the patch removal. At concentrations of 50, 25 and 10% necrosis appeared on the place of intra dermal injections 24 h after the patch removal. At concentration of 5% very slight erythema was observed in both animals one hour after the patch removal. Based on the results, the test substance at concentration of 5% was used for the intra-dermal treatment in the main study.

For the dermal application, 0.5 mL test substance at concentrations of 25, 50, 75% (w/v) and in undiluted state were applied onto the clipped and shaved skin of the animals. It was found that 0.5 mL of test formulation at concentrations of 25, 50, 75% (w/v) and in undiluted state produced no reaction on the skin of guinea pigs. For the dermal application of challenge treatment, the test substance was used at concentrations of 25, 50, 75% (w/v) and in undiluted state.

From the preliminary study results, test substance was used in undiluted state for dermal induction treatment. Before the dermal exposure the test area was painted with 0.5 mL of 10% sodium dodecyl sulphate in vaseline 24 h prior topical induction application, in order to create a local irritation. For the challenge treatment the test substance was used in undiluted state because primary irritation sign was not found at this concentration during the preliminary dose range finding study.

- Main study:
- Intra-dermal induction exposure:
Before starting the exposure an area of approximately 5x5 cm on the scapular region of animals was clipped free of hair and shaven close with care.
Intra-dermal treatment:
Test groups: A row of three injections, six in all, was made on each side of test animals, as follows:
2 injections with 0.10 mL of Freund's Adjuvant mixed with physiological saline, (1:1 v/v),
2 injections with 0.10 mL of the test substance (5%) homogenized sunflower oil,
2 injections with 0.10 mL of test substance (5%), formulated in a 1:1 (v/v) mixture of Freund's Adjuvant and sunflower oil

- Control group:
The control animals were treated similarly as the test group however, the vehicle, without the test substance was used for injections, as follows:
2 injections with 0.1 mL mix of Freund's Adjuvant and physiological saline (1:1) (v/v),
2 injections with 0.1 mL of sunflower oil,
2 injections with 0.1 mL of 50% (w/v) physiological saline, in a 1:1 mixture (v/v) Freund's Adjuvant and sunflower oil.

- Dermal induction exposure:
Seven days after the intra-dermal injections, the test animals were exposed to test substance, whereas the control animals were treated with sunflower oil, as vehicle. Closed patch was applied in the following manner: in case of the test animals 0.5 mL of test substance (at concentration of 100%) was spread on the surface prepared previously and covered with a standard (5x5 cm) size of porous gauze patch. Control animals were treated dermally with 0.5 mL of vehicle and the dressing was prepared and applied as for the test animals. The exposed areas were covered for 48 h with porous gauze fastened with "Leucoplast" (Closed Patch Test).

- Challenge exposure:
Two weeks after the dermal treatment the animals were exposed to the challenge dose, dermally. 24 h before the challenge treatment the left and the right flank areas (5x5 cm) of each animal were prepared for application. The challenge was performed as a dermal exposure (closed patch test). Left shaved flank areas of the animals (both the test and the control) were treated with 0.5 mL of the test substance (in undiluted state). The right shaved flank areas were treated with 0.5 mL of vehicle. Time of exposure was 24 h. After the challenge exposure, the bandages were removed and the dermal responses were examined and scored. The first scoring was performed 48 h post applications (24 h value). The second grading was carried out the day thereafter (48 h value).

- OBSERVATIONS:
- Mortality: Animal were observed daily from delivery of the animals to the termination of the experiment.
- Clinical signs: daily during the study.
- Body weight: The body weights of individual animals were recorded at the beginning and at the end of experiments.
- Skin reactions were observed and recorded at:
Intra-dermal induction exposure: 24 h after the treatment,
Dermal induction exposure: 1, 24, 48 and 72 h after the patch removal,
Challenge exposure: 24 and 48 h after the patch removal.
- The dermal irritation scores (in case of the preliminary study (primary irritation) and in cases of induction dermal exposures) were evaluated according to the scoring system by Draize (1977). Classification and scoring (after challenge exposure) was done according to the test guidelines.

Challenge controls:

Control animals received vehicle only.

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole tested in another study

Results and discussion

Positive control results:

The sensitivity and reliability of the experimental procedure is assessed twice in a year by use of reference substances which are known to have moderate skin sensitisation properties such as 2-Mercaptobenzothiazole. The reference substance 2-Mercaptobenzothiazole caused positive responses in 40% of the treatment group animals, thus confirming sensitivity and reliability of the experimental technique used.

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 2

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Results 3

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Any other information on results incl. tables

- Mortality: No deaths occurred in the study

- Very slight but well defined erythema in test group animals and very slight erythema in control group animals were observed post intra dermal induction exposure.

- One hour post the patch removal in the test and control animals, very slight to moderate erythema and in some cases very slight oedema was found on the treated skin surface. 24 h after the patch removal in six animals of test group and in one animal of control group primary irritation signs were not found. 72 h after the patch removal all animals were symptom-free.

- After the challenge exposure with test substance in undiluted state, no positive response was observed in test and control animals at 24 and 48 h post patch removal.

- Body weight: There were no notable differences between the test animal group and the control group.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Conclusions:

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

Executive summary:

A guinea pig maximization study was conducted to evaluate the skin sensitisation potential of the test substance according to OECD Guideline 406, EPA OPPTS 870.2600 and EU Method B.6, in compliance with GLP. Based on the dose range finding study, a 5% concentration was selected for intradermal induction and 100% (undiluted) was selected for topical induction and challenge exposure. In the main test, 30 female guinea pigs were randomised into a control group containing 10 animals and a treatment groups containing 20 animals. Sunflower oil was used as vehicle. Two weeks after the last induction exposure, a challenge dose (dermal application in undiluted state) was administered to check elicitation. Ten control guinea pigs were simultaneously exposed to vehicle during the induction phase and treated with the test substance (undiluted) only for the challenge (elicitation) phase. No signs of contact sensitisation were detected in guinea pigs previously exposed to the test substance. There were no positive responses post challenge in any group. There was no mortality and no significant differences in body weight gain between the test animal group and the control group were recorded.  In a separate trial, the reference substance 2 -mercaptobenzothiazole caused positive responses in 40% of the treatment group animals, thus confirming sensitivity and reliability of the experimental technique used. The net response value in the present study represented an incidence rate of 0% and the net score value of 0.00. Under the study conditions, the test substance was not considered to be a skin sensitiser (Stahl, 2016).