Reaction mass of N,N’-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide), Octadecanamide, 12-hydroxy-N-[2-[(1-oxooctadecyl)amino]ethyl]- and Octadecanoic acid, 12-hydroxy-, 1-hexyl-12-[[2-[(12-hydroxy-1-oxooctadecyl)amino]ethyl]amino]-12-oxododecyl ester

Endpoint summary

Description of key information

A guinea pig maximization study was conducted to evaluate the skin sensitization potential of the read-across substance bisamide (UVCB) according to OECD Guideline 406 and EU Method B.6 in compliance with GLP. During the induction phase, 20 guinea pigs (15 males and 15 females) were exposed to 5% test substance in corn oil via intra-dermal injection and 25% test substance in ethanol/drinking water (80/20) via 48 h topical occlusive patches. During the induction phase, exposure occurred via 10% (in acetone) 24 h topical occlusive patches. No unscheduled deaths, changes in body weight or any clinical signs were observed during the study. Scabs and cracks were seen in all animals, associated with wounds in some of them. No cutaneous reactions were observed in the control group at the 24 and 48 h readings. Further, at the 24 h reading in the test substance-treated group, a discrete or moderate erythema, associated with dryness of the skin, was noted on the right flank (treated with test substance) of 13/20 animals. At the 48 h reading, a discrete or moderate erythema was observed on the right flank (treated with test substance) of 14/20 animals, associated with dryness of the skin in six of them and edema in four of them. In addition, at the 24 and 48 h readings, an intense erythema, associated with edema and dryness of the skin, was observed on the right flank of 1/20 animals. Overall, the cutaneous reactions on the right flank of the test substance-treated group were of higher incidence and severity than those recorded on the right flank of the control group during the challenge phase. Therefore, these findings were considered to be attributed to delayed contact hypersensitivity. Under the conditions of the study, the test substance was considered to induce delayed contact hypersensitivity in 15/20 (75%) guinea pigs.

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 02 August 2012 to 17 September 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Justification for non-LLNA method:

In the context of its REACH registration, 12-HSA was tested for sensitization in a Local Lymph Node Assay (LLNA). The study produced a positive response (Sanders A., 2012). For the following reasons, the response seen in the LLNA study with 12-HSA was considered as a false positive:
- From a structural point of view, 12-HSA does not contain electrophilic groups that could potentially trigger skin sensitization reactions. (Q)SAR analysis suggests that 12-HSA is not a sensitizer,
- 12-HSA has a history of safe use in consumer products with substantial skin contact (e.g. cosmetics, laundry and cleaning products), with no evidence of skin sensitization issues. Furthermore, experience from worker exposure to 12-HSA at both production and handling sites suggests that the substance does not cause sensitizing effects,
- During the validation of the LLNA study, the observation was made that it often overestimates the sensitization potential for some substances, e.g. surfactants, fatty acids, fatty alcohols and siloxanes (Ball et al., 2011). Testing conducted with a number of fatty acids showed that some cause positive effects in the LLNA test but are then negative in the GMPT study. The LLNA results are therefore considered to be false positives. This is the case for 12-HSA which was recently tested in a GPMT study.

For the above mentioned reasons, it was advisable to conduct a Guinea Pig Maximisation Test rather than a Local Lymph Nodes Assay for assessing the skin sensitization potential of the test substance.

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 360 g (range: 284 g to 393 g) and the females had a mean body weight of 352 g (range: 276 g to 382 g)
- Fasting period before study: No
- Housing: 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: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 07 August 2012 to 17 September 2012.

Route:

intradermal and epicutaneous

Vehicle:

other: Corn oil for intradermal induction injections; ethanol/water for topical induction dose and acetone for topical challenge dose

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: 10%**
** highest non-irritant concentration based on preliminary assays

Route:

epicutaneous, occlusive

Vehicle:

other: Corn oil for intradermal induction injections; ethanol/water for topical induction dose and acetone for topical challenge dose

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: 10%**
** highest non-irritant concentration based on preliminary assays

No. of animals per dose:

Main test: 10 control animals and 20 treated animals (15 males and 15 females).

Details on study design:

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: Topical induction: 48 h
- Site: Interscapular region
- Dose: Intradermal injection: On Day 1, 3 pairs (top, middle and down) of 5% test substance in corn oil were administered by intradermal injection in the clipped interscapular region using a sterile plastic syringe fitted with a sterile single use needle. Topical induction: On Day 8, a filter paper (approximately 8 cm2; held in place with occlusive dressing) was fully-loaded with 25% test substance in ethanol/drinking water treated by reverse osmosis (80/20), and then applied to the clipped interscapular region, over the intradermal injection sites for 48 h. The induction phase was followed by a 14-d rest period.
- Frequency of applications: Once intradermal, once topical
- Type of patch: Occlusive patch (i.e., a filter paper (approximately 8 cm2) fully-loaded with the dose formulations was held in place by means of an occlusive dressing).
- Observations: The cutaneous reactions were evaluated before treatment, 24 and 48 h after removal of the dressing.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 22
- Exposure period: 24 h
- Dose: On Day 22, a Finn Chamber filter paper (held in place with occlusive dressing) was fully-loaded with the 10% test substance in acetone, and then applied to the clipped interscapular region, over the intradermal injection sites for 24 h. The filter paper was held in place by means of an occlusive dressing for 48 hours..
- Site: Right flank (test substance) and left flank (vehicle)
- Type of patch: Occlusive patch (i.e., a Finn Chamber® filter paper fully-loaded with the dose formulations was held in place by means of an occlusive dressing).
- Observations: The cutaneous reactions were evaluated before treatment, 24, 48 h after removal of dressing.


OBSERVATIONS:
- Morbidity and mortality: Each animal was checked for mortality and morbidity at least once a day during the treatment and observation periods, including weekends and public holidays.
- Clinical signs: Each animal was observed at least once a day, at approximately the same time, for the recording of clinical signs.
- Cutaneous reactions: Cutaneous reactions were scored in each animal according to the following scale:
. no visible change ...............................................................................................................................0
. discrete or patchy erythema ..............................................................................................................1
. moderate and confluent erythema.....................................................................................................2
. intense erythema ...............................................................................................................................3

Any observations of edema or other lesions were recorded.
- Body weight: The body weight of each animal was recorded the day of group allocation, then on the first day of treatment and at sacrifice.

Challenge controls:

Control animals received vehicle only.

Positive control substance(s):

not required

Remarks:

mercaptobenzothiazole tested in another study

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5% intradermal injection

No. with + reactions:

13

Total no. in group:

20

Clinical observations:

discrete or moderate erythema, associated with dryness of the skin

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5% intradermal injection. No with. + reactions: 13.0. Total no. in groups: 20.0. Clinical observations: discrete or moderate erythema, associated with dryness of the skin.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5% intradermal injection

No. with + reactions:

14

Total no. in group:

20

Clinical observations:

discrete or moderate erythema, associated with dryness of the skin and edema

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5% intradermal injection. No with. + reactions: 14.0. Total no. in groups: 20.0. Clinical observations: discrete or moderate erythema, associated with dryness of the skin and edema.

- Mortality: No unscheduled deaths occurred.

- Clinical signs: No clinical signs were observed in any animals. Scabs and cracks were observed in all groups during the observation period, associated with wounds in some of them.

- Cutaneous reactions:

- In the control group, at the 24- and 48-h readings, no cutaneous reactions were observed on the left flank (treated with vehicle) and on the right flank (treated with test substance) of animals.

- In the test substance-treated group: At the 24-h reading: A discrete erythema (grade 1) was noted on the left flank (treated with the vehicle) of 1/20 animals. A discrete or moderate erythema (grade 1 or 2), associated with dryness of the skin, was observed on the right flank (treated with test substance) of 13/20 animals. At the 48-h reading, a discrete or moderate erythema (grade 1 or 2) was observed on the right flank (treated with test substance) of 14/20 animals, associated with dryness of the skin in six of them and edema in four of them.

In addition, at the 24- and 48-h readings, an intense erythema (grade 3), associated with edema and dryness of the skin, was observed on the right flank of one male.

The cutaneous reactions observed on the right flank (test substance-treated) of the test substance-treated group were of higher incidence and severity than those recorded on the right flank (test substance-treated during challenge phase) of the control group. Therefore, these findings were considered to be attributed to delayed contact hypersensitivity.

- Body weight: The body weight change of the test substance-treated animals was similar to that of controls.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the conditions of the study, the test substance was considered to induce delayed contact hypersensitivity in 15/20 (75%) guinea pigs.

Executive summary:

A guinea pig maximization study was conducted to evaluate the skin sensitization potential of the test substance according to OECD Guideline 406 and EU Method B.6 in compliance with GLP.

During the induction phase, 20 guinea pigs (15 males and 15 females) were exposed to 5% test substance in corn oil via interdermal injection and 25% test substance in ethanol/drinking water (80/20) via 48 h topical occlusive patches. During the induction phase, exposure occurred via 10% (in acetone) 24 h topical occlusive patches. No unscheduled deaths, changes in body weight or any clinical signs were observed during the study. Scabs and cracks were seen in all animals, associated with wounds in some of them. No cutaneous reactions were observed in the control group at the 24 and 48 h readings. Further, at the 24 h reading in the test substance-treated group, a discrete or moderate erythema, associated with dryness of the skin, was noted on the right flank (treated with test substance) of 13/20 animals. At the 48 h reading, a discrete or moderate erythema was observed on the right flank (treated with test substance) of 14/20 animals, associated with dryness of the skin in six of them and edema in four of them. In addition, at the 24 and 48 h readings, an intense erythema, associated with edema and dryness of the skin, was observed on the right flank of 1/20 animals. Overall, the cutaneous reactions on the right flank of the test substance-treated group were of higher incidence and severity than those recorded on the right flank of the control group during the challenge phase. Therefore, these findings were considered to be attributed to delayed contact hypersensitivity.

Under the conditions of the study, the test substance was considered to induce delayed contact hypersensitivity in 15/20 (75%) guinea pigs.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

No information is available on the respiratory sensitization potential of bisamide (multi). However, based on data from the read-across substance bisamide (UVCB), the substance is expected to have low vapour pressure (0.0001 Pa at 25°C) so that normal processing and use conditions will not generate inhalation exposure. In case dust, aerosols or vapours are created under particular conditions, appropriate risk management measures such as closed systems, exhaust ventilation or wearing of respirators are implemented to control exposure. Respiratory irritation is therefore not expected to pose an issue for human health.

Justification for classification or non-classification

According to the results of an in vivo guinea pig maximization study on the read-across substance bisamide (UVCB), bisamide (multi) qualifies for classification as Skin Sens. 1 (sub-category 1B) - H317 (May cause allergic skin reactions).