Reaction products of 12-hydroxyoctadecanoic acid with ethane-1,2-diamine and hexane-1,6-diamine and 1,3-phenylenedimethanamine

Endpoint summary

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:

From 2012-08-19 to 2012-10-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

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)

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: breeder: Charles River Laboratories France, L’Arbresle, France
- Age at study initiation: approximately 1 to 2 months old on the day of treatment
- Mean body weight at study initiation: the males had a mean body weight of 329 g (range: 284 g to 527 g) and the females had a mean body weight of 307 g (range: 262 g to 385 g)
- Fasting period before study: no
- Housing: individually housed in polycarbonate cages with stainless steel lid
- Diet: 106 pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 5 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 11 September 2012 to 22 October 2012.

Route:

intradermal and epicutaneous

Vehicle:

other: Corn oil for intradermal injection, Ethanol/drinking water treated by reverse osmosis (80/20) for induction application and acetone for challenge application

Concentration / amount:

Induction phase:
Concentration for intradermal injection: 5% *
Concentration for topical application: 25% *
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Challenge phase:
Concentration for topical application:25%**
** highest non-irritant concentration based on preliminary assays

Route:

epicutaneous, occlusive

Vehicle:

other: Corn oil for intradermal injection, Ethanol/drinking water treated by reverse osmosis (80/20) for induction application and acetone for challenge application

Concentration / amount:

Induction phase:
Concentration for intradermal injection: 5% *
Concentration for topical application: 25% *
*: highest to cause mild-to-moderate skin irritation based on preliminary assays

Challenge phase:
Concentration for topical application:25%**
** highest non-irritant concentration based on preliminary assays

No. of animals per dose:

- preliminary test: 4 treated animals
- main test: 10 control animals and 20 treated animals

Details on study design:

RANGE FINDING TESTS: conducted to define the concentrations to be tested in the main study

-Intradermal exposure:
Hair over the scapulae was removed using electric clippers before treatment.
Intradermal administration of 0.1 ml of the test material (TM) at increasing concentrations. An homogenous suspension was obtained at the concentration of 25% in corn oil but the dosage form preparation did not pass into the dermis at this concentration. Therefore concentrations of 10% and 5% in corn oil were tested in 2 animals in duplicate.
Evaluation of the potential cutaneous reactions: 24, 48 hours and 6 days after injection

- Epicutaneous exposure:
Hair over the scapulae was removed using electric clippers before treatment.
Each selected concentration applied to a filter paper patch measuring 8cm2 for 48 hours under occlusive dressing . 2 concentrations (25% and 10% w/v in ethanol/drinking water treated by reverse osmosis (80/20) were tested in 2 animals.
Evaluation of the potential cutaneous reactions 24 and 48 hours after patch removal.


MAIN STUDY

A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal, 1 epicutaneous
- Exposure period: epicutaneous: 48 hours

-> TEST GROUPS:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with sterile water
. TM at 0.5 % w/v in vehicle
. TM at 0.5% w/v in a 50/50 (v/v) mixture of FCA and sterile water

Epicutaneous exposure
Application of 8 cm2 patch saturated with the undiluted TM to the scapular region and held in place for 48 hours using an occlusive dressing.

-> CONTROL GROUP:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with sterile water
. vehicle
. A mixture 50/50 (v/v) FCA diluted to 50% with sterile water, and vehicle

Epicutaneous exposure
Application of 8 cm2 patch saturated with the vehicle to the scapular region and held in place for 48 hours using an occlusive dressing.

- Site:
Intradermal exposure
6 injections in the clipped area (3x 4cm) in the scapular region

Epicutaneous exposure
3x 4cm area over the scapulae

- Frequency of applications:
One intradermal injection and one epicutaneous application on Day 8 on the same site.

- Duration:
Epicutaneous exposure: 48 hours

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 22
- Exposure period: 24 hours

-> TEST GROUPS:
Challenge: TM at 25% in the vehicle on the right flank and vehicule on the left flank (occlusive epicutaneous application)

-> CONTROL GROUPS:
Same treatment as test group
- Site: posterior flanks
- Evaluation (hr after challenge): 24 , 48 hours after removal of the dressing according to the method of Draize.

OTHER:
As in the preliminary test, the highest well-tolerated concentration was shown to be non irritant after topical application, 0.5 mL of sodium lauryl sulfate at the concentration of 10% (w/w) in Vaseline was applied to the induction site on day 7 (all animals) in order to induce a local irritation.

Challenge controls:

Yes - see above.

Positive control substance(s):

yes

Remarks:

mercaptobenzothiazole

Positive control results:

Regular checks were performed at the laboratory with the positive control substances benzothiazole.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

25%

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: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

25%

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: 25%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

4

Total no. in group:

20

Clinical observations:

Erythema grade 1 in 4 animals

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 4.0. Total no. in groups: 20.0. Clinical observations: Erythema grade 1 in 4 animals.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25%

No. with + reactions:

5

Total no. in group:

20

Clinical observations:

Erythema grade 2 in 1 animal; Erythema grade 1 in 4 animals + skin dryness

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 5.0. Total no. in groups: 20.0. Clinical observations: Erythema grade 2 in 1 animal; Erythema grade 1 in 4 animals + skin dryness.

The preliminary study indicated that the test material injected at 5% concentration was reasonably well tolerated. A concentration of 25% was selected for both the topical induction and the challenge phase as it was judged to be non-irritant.

Table 7.4.1/1 Individual skin reactions after challenge

Group	Sex	Animal number	Skin reactions (Hour after removal of dressings)
			24 hours		48 hours
			LF	RF	LF	RF
Control group	Male	Y40043	0	0	0	0
		Y40044	0	0	0	0
		Y40045	0	0	0	0
		Y40046	0	0	0	0
		Y40047	0	0	0	0
	Female	Y40093	0	0	0	0
		Y40094	0	0	0	0
		Y40095	0	0	0	0
		Y40096	0	0	0	0
		Y40097	0	0	0	0
Treated group	Male	Y40048	0	0	0	0
		Y40049	0	1	0	2/S
		Y40050	0	0	0	0
		Y40051	0	0	0	0
		Y40052	0	0	0	0
		Y40053	0	0	0	1
		Y40054	0	1	0	0
		Y40055	0	0	0	0
		Y40056	0	1	0	1
		Y40057	0	0	0	1/S
	Female	Y40098	0	0	0	0
		Y40099	0	0	0	0
		Y40100	0	0	0	0
		Y40101	0	1	0	1/S
		Y40102	0	0	0	0
		Y40103	0	0	0	0
		Y40104	0	0	0	0
		Y40105	0	0	0	0
		Y40106	0	0	0	0
		Y40107	0	0	0	0

LF = Left flank (vehicle)

RF = Right flank (test item at the concentration of 25%)

S= Dryness of the skin

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The substance induced delayed contact hypersensitivity in 5/20 (25%) guinea pigs and was considered as a mild sensitizer.
According to Regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures, the substance is not classified as the percentage of animals showing positive responses is lower than 30%.

Executive summary:

The potential of the substance to induce delayed contact hypersensitivity was assessed in guinea pigs according to OECD guideline 406 and in compliance with Good Laboratory practices.

The induction phase was realized both by intradermal route on day 1 (Test material 5% in corn oil) and by cutaneous route on day 8 (Test material 25% in ethanol/drinking water treated by reverse osmosis 80/20) in 2 groups of guinea pigs: 10 for control group and 20 for treated group. As in the preliminary test, the highest well-tolerated concentration was shown to be non irritant after topical application, sodium lauryl sulfate 10% in Vaseline was applied to the induction site on day 7 in order to induce a local irritation.The challenge phase was realized on day 22 by cutaneous application of the test material at 25% in acetone. The cutaneous reactions were scored 24 and 48 after the challenge phase.

Scabs were observed in all animals during the observation period, associated with cracks in some of them. As these skin reactions were noted at the interscapular region, they were attributed to the treatment route and irritant properties of the test item at the concentration used for the intradermal injection. In the control group, at the 24- and 48-hour readings, no cutaneous reactions were observed. In the treated group, a discrete erythema (grade 1) was noted in 4 /20 animals at the 24-hour reading

At the 48-hour reading, a discrete to moderate erythema (grade 1 to 2) was observed in 5/20 animals, associated with dryness of the skin for 3 of them.

The persistent cutaneous reactions observed in 5/20 animals of the test item-treated group were attributed to delayed contact hypersensitivity. It was concluded that the substance was a mild sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The potential of the substance to induce delayed contact hypersensitivity was assessed in guinea pigs according to OECD guideline 406 and in compliance with Good Laboratory practices.

The induction phase was realized both by intradermal route on day 1 (Test material 5% in corn oil) and by cutaneous route on day 8 (Test material 25% in ethanol/drinking water treated by reverse osmosis 80/20) in 2 groups of guinea pigs: 10 for control group and 20 for treated group. As in the preliminary test, the highest well-tolerated concentration was shown to be non irritant after topical application, sodium lauryl sulfate 10% in Vaseline was applied to the induction site on day 7 in order to induce a local irritation.The challenge phase was realized on day 22 by cutaneous application of the test material at 25% in acetone. The cutaneous reactions were scored 24 and 48 after the challenge phase.

Scabs were observed in all animals during the observation period, associated with cracks in some of them. As these skin reactions were noted at the interscapular region, they were attributed to the treatment route and irritant properties of the test item at the concentration used for the intradermal injection.

In the control group, at the 24- and 48-hour readings, no cutaneous reactions were observed. In the treated group, a discrete erythema (grade 1) was noted in 4 /20 animals at the 24-hour reading.

At the 48-hour reading, a discrete to moderate erythema (grade 1 to 2) was observed in 5/20 animals, associated with dryness of the skin for 3 of them.

The persistent cutaneous reactions observed in 5/20 animals of the test item-treated group were attributed to delayed contact hypersensitivity. It was concluded that the test item was a mild sensitizer (Gerbeix, 2013b).

Migrated from Short description of key information:
The potential of the substance to induce delayed contact hypersensitivity was investigated using the Maximization method of Magnusson and Kligman (OECD 406, GLP) . it was advisable to conduct a Guinea Pig Maximisation Test rather than a Local Lymph Nodes Assay because the substance contained an impurity which was known to give false positive result in the Local Lymph Nodes Assay.

Justification for selection of skin sensitisation endpoint:
Study performed according to the OECD guideline 406 and GLP compliant

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance induced delayed contact hypersensitivity in 5/20 (25%) guinea pigs and was considered as a mild sensitizer. According to Regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures, the substance is not classified as the percentage of animals showing positive responses is lower than 30%.