Registration Dossier

Toxicological information

Endpoint summary

Currently viewing:

Administrative data

Description of key information

The substance is not a skin sensitiser, based on the results of a study performed according to OECD TG 406.

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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The decision to perform this study in preference to the LLNA was taken based on the surface active properties of the substance. The potential for surface active substances to demonstrate sensitizing properties has been shown to be more accurately predicted using the M&K procedure than the LLNA. Some surfactants have been shown to demonstrate false positive responses in the LLNA, for example sodium lauryl sulfate.
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L’Arbresle, France.
- Age at study initiation: 1 to 2 months old
- Weight at study initiation: males mean body weight 368 g (range: 279 g to 418 g); females mean body weight 346 g (range: 276 g to 414 g).
- Housing: individually housed in polycarbonate cages with stainless steel lid (Tecniplast 2154, 940 cm²) containing autoclaved sawdust (SICSA, Alfortville, France).
- Diet: ad libitum - 106 pelleted maintenance diet, batch No. 12010 (SAFE, Augy, France)
- Water: ad libitum - tap water (filtered with a 0.22 μm filter)
- Acclimation period: At least 5 days

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

IN-LIFE DATES: From: To: 13 July - 07 September 2012
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
Preliminary test:
Induction: Intradermal injection 0.25, 0.5, 1, 2.5, 5 or 10 %; topical application 1, 2.5, 5 or 10 %
Main test:
Induction: Intradermal injection 1 %; Topical application 10 %
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
Preliminary test:
Challenge: Topical application 5 or 10 %
Main test:
Challenge: Topical application 1 %
No. of animals per dose:
Control group: 5 males + 5 females
Test group: 10 males + 10 females
Details on study design:
RANGE FINDING TESTS:see below

MAIN STUDY

A. INDUCTION EXPOSURE

STAGE 1 - INTRACUTANEOUS
- No. of exposures: 1
- Exposure period: day 1
- Test groups: FCA/0.9% NaCl; 1% test substance; 1% test substance in FCA/0.9% NaCl
- Control group: FCA/0.9% NaCl; 0.9% NaCl; 0.9% NaCl in FCA/0.9% NaCl
- Site:each side of the midline
- Frequency of applications: single application
- Duration: -
- Concentrations: see above

STAGE 2 - TOPICAL
- No. of exposures: 1
- Exposure period: day 8
- Test groups: 10 % substance
- Control group: 0 % substance
- Site: Back
- Frequency of applications: single application
- Duration: -
- Concentrations: see above
- OTHER: On day 7 0.5 mL of sodium lauryl sulfate at a concentration of 10 %w/w in vaseline was applied to the induction site in order to induce a local irritation

B. CHALLENGE EXPOSURE
- No. of exposures:1
- Day(s) of challenge: 22
- Exposure period:-
- Test groups: 1 %
- Control group: 0 %
- Site: back
- Concentrations:see above
- Evaluation (hr after challenge): 24 & 48 h
Challenge controls:
Treated with 1 % substance on right flank
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: vehicle control
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: vehicle control
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
1 %
No. with + reactions:
2
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
1 %
No. with + reactions:
3
Total no. in group:
20
Group:
positive control
Remarks on result:
not measured/tested

Preliminary test:

Mortality: No unscheduled deaths occurred during the study

Clinical signs: No clinical signs in any animals

Cutaneous reactions:

By intradermal injections

The dosage form preparation did not pass into the dermis at the concentrations of 10% (w/w). Discrete to moderate erythema (grade 1 to 2) were observed in the interscapular region of animals treated at 2.5 and 5% with and/or without FCA all over the observation period. Necrosis was noted on the injection sites of all animals at the 48-hour reading and/or on day 7. As a result, in order to respect the criteria for the selection of concentrations described in the OECD 406, an additional preliminary assay was performed using the intradermal route.

An irritation was observed in the interscapular region of animals treated at 0.25, 0.5 and 1% with and/or without FCA at the 24 and 48 hours observation times. On day 7, no erythema was noted in the interscapular region of all animals in the absence of FCA, except

female No. Y40333 treated at the concentration of 1%. In presence of FCA, discrete to intense erythema (grade 1 to 3) was noted in all animals, associated with scabs at some injection sites. No necrosis was noted on the injection sites of all animals all over the observation.

Therefore, in order to respect the criteria for the selection of concentrations described in the OECD 406, the concentration chosen for intradermal injections of the main test was 1%.

By topical application

Under conditions of induction phase

No irritation was observed in both animals treated at the concentrations of 10 and 5%. A dryness of the skin was noted in the female (No. Y40332) treated at the concentration of 10% only. Therefore, in order to respect the criteria for the selection of concentrations described in the OECD 406, the concentration chosen for the topical application of the induction phase (day 8) was 10%.

Under conditions of challenge phase

A discrete or moderate erythema (grade 1 or 2) was observed at the concentrations of 5 and 10% at the 48-hour reading (except on the left flank of male Y40301 treated at the concentration of 5%). Therefore, in order to respect the criteria for the selection of concentrations described in the OECD 406, an additional preliminary assay was performed under the challenge conditions. A discrete erythema (grade 1) was observed at the concentrations of 2.5 at the 24- and 48-hour readings, associated with dryness of the skin at the end of the observation period. No irritation was noted at the concentrations of 1%. Therefore, in order to respect the criteria for the selection of concentrations described in the OECD 406, the concentration chosen for the challenge application (day 22) was 1%.

Body weight: The body weight of the animals was unaffected by the test item treatment.

Main test:

Mortality: No unscheduled deaths occurred during the main test.

Clinical signs: No clinical signs indicative of systemic toxicity were observed in any animals. Scabs and cracks were observed in all groups 4 and 5 animals during the observation period, associated with wounds in some of them.

Cutaneous reactions:

In the control group, at the 24-hour reading, a discrete erythema (grade 1) was observed on the right flank (treated with test item) of 2/10 animals (Y40307 and Y40308). At the 48-hour reading, a discrete erythema (grade 1) was observed on the left flank (treated with vehicle) of 1/10 animals (Y40336) and on the right flank (treated with test item) of 2/10 animals (Y40308 and Y40336). In the test item-treated group, at the 24-hour reading, a discrete (grade 1) was noted at left flank (treated with vehicle) of 1/20 animals (Y40316). A discrete to moderate erythema (grade 1 to 2) was noted on the right flank (treated with the test item) of 5/20 animals. A skin dryness was also noted on the right flank (treated with the test item) in 6/20 animals. At the 48-hour reading, a discrete erythema (grade 1) was observed at left flank (treated with vehicle) of 1/20 animals (Y40311). A discrete to intense erythema (grade 1 to 3) was noted at right flank (treated with test item) of 5/20 animals, associated with edema in two of them. A skin dryness was also noted on the right flank (treated with the test item) in 6/20 animals. The cutaneous reactions (grade 1) observed on the right flank treated with the test item of control animals were considered to be attributed to the irritant properties of the test item. Therefore, the discrete erythema observed on the right flank of the test item-treated animals being of similar incidence than in the control group, these reactions were not considered in the evaluation of the results. The cutaneous reactions (grade 2 to 3) observed on the right flank (treated with the test item) of test item-treated animals (group 5) were considered to be of higher incidence and severity than those recorded at the right flank (treated with the test item) of control animals (group 4). As the moderate erythema (grade 2) noted at the 24-hour reading on the right flank (treated with the test item) of animal Y40345 became a discrete erythema (grade 1) at the 48-hour reading, it was not considered to be attributed to contact delay hypersensitivity. However, the other findings noted in the group 5 animals were considered to be attributed to delayed contact hypersensitivity.

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

Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitisation potential of the substance was assesses in a study performed according to OECD TG 406 (Magnusson and Kligman Maximisation Test) under GLP using Hartley Crl: (HA) BR guinea pigs [CiToxLAB (2012)]. The decision to perform this study in preference to the LLNA was taken based on the surface active properties of the substance. The potential for surface active substances to demonstrate sensitizing properties has been shown to be more accurately predicted using the M&K procedure than the LLNA. Some surfactants have been shown to demonstrate false positive responses in the LLNA, for example sodium lauryl sulfate.

A preliminary study was performed to determine the appropriate dose level of substance to use in the main study following intradermal and topical administration. Six concentrations of the substance in 0.9% saline solution were tested by intradermal injection using 2 male and 2 female animals. The dose formulations with or without Freund’s Complete Adjuvant were administered by intradermal injection in the clipped interscapular region using a sterile plastic syringe fitted with a sterile single use needle. A constant dosage-volume of 0.1 mL/injection was used. Discrete to moderate erythema (grade 1 to 2) were observed in the interscapular region of animals treated at 2.5 and 5% with and/or without FCA all over the observation period. Necrosis was noted on the injection sites of all animals at the 48-hour reading and/or on day 7. As a result, in order to respect the criteria for the selection of concentrations described in the OECD 406, an additional preliminary assay was performed using the intradermal route. An irritation was observed in the interscapular region of animals treated at 0.25, 0.5 and 1% with and/or without FCA at the 24 and 48 hours observation times. On day 7, no erythema was noted in the interscapular region of all animals in the absence of FCA, except for one female treated at the concentration of 1%. In presence of FCA, discrete to intense erythema (grade 1 to 3) was noted in all animals, associated with scabs at some injection sites. No necrosis was noted on the injection sites of all animals all over the observation. Therefore, in order to respect the criteria for the selection of concentrations described in the OECD 406, the concentration chosen for intradermal injections of the main test was 1%. 

For topical induction, a filter paper (approximately 8 cm2) was fully-loaded with the dose formulations, and then applied to the clipped interscapular region of two animals. The filter paper was held in place by means of an occlusive dressing for 48 hours. On removal of the dressing, no residual test item was observed. Cutaneous reactions were evaluated before treatment, 24 and 48 hours after removal of the dressing. No irritation was observed in both animals treated at concentrations of 10 and 5%. A dryness of the skin was noted in the female treated at the concentration of 10% only. Therefore, in order to respect the criteria for the selection of concentrations described in the OECD 406, the concentration chosen for the topical application of the induction phase (day 8) was 10%. For topical challenge, a Finn Chamber® filter paper was fully-loaded with the dosage forms, and then applied to the shaved posterior right or left flank of two animals. The chamber was held in place by means of an occlusive dressing for 24 hours. On removal of the dressing, no residual test item was observed. Cutaneous reactions were evaluated before treatment, 24 and 48 hours after removal of the dressing. The test concentrations were adapted until the maximum non-irritant concentration was determined. A discrete or moderate erythema (grade 1 or 2) was observed at the concentrations of 5 and 10% at the 48-hour reading (except on the left flank of the male treated at the concentration of 5%). Therefore, in order to respect the criteria for the selection of concentrations described in the OECD 406, an additional preliminary assay was performed under the challenge conditions. A discrete erythema (grade 1) was observed at the concentrations of 2.5 at the 24- and 48-hour readings, associated with dryness of the skin at the end of the observation period. No irritation was noted at the concentrations of 1%. Therefore, in order to respect the criteria for the selection of concentrations described in the OECD 406, the concentration chosen for the challenge application (day 22) was 1%.

The main study was performed using 20 animals and a further 10 animals were used as vehicle (tap water) controls. For the intradermal induction the animals received three pairs of injections (0.1 mL each) as follows: Freund’s complete adjuvant (diluted 1:1 with 0.9 % NaCl); 1 % substance solution; 1 % substance solution in a 1:1 mixture with FCA/0.9 % NaCl solution. The first and second pairs of injections were given close to each other and nearest the head, while the third pair was given towards the caudal part of the test area. On Day 7, prior to topical induction, the area was shaved again and the skin coated within sodium lauryl sulfate (0.5 mL, 10 % solution in vaseline) in order to ensure the induction of local irritation. On Day 8 the animals were exposed topically under occlusive conditions to a 10 % solution of the substance for 48 hours. Control animals received the vehicle only. The induction phase was followed by a 14-day rest period. At the end of this period the test animals and control animals were challenged with a 1 % solution of substance for 24 hours under occlusive conditions. Dermal reactions were evaluated before treatment and 24 and 48 hours after removal of the dressing.

In the control group, at the 24-hour reading, a discrete erythema (grade 1) was observed on the right flank (treated with test item) of 2/10 animals. At the 48-hour reading, a discrete erythema (grade 1) was observed on the left flank (treated with vehicle) of 1/10 animals and on the right flank (treated with test item) of 2/10 animals. In the test item-treated group, at the 24-hour reading, a discrete (grade 1) was noted at left flank (treated with vehicle) of 1/20 animals. A discrete to moderate erythema (grade 1 to 2) was noted on the right flank (treated with the test item) of 5/20 animals. A skin dryness was also noted on the right flank (treated with the test item) in 6/20 animals. At the 48-hour reading, a discrete erythema (grade 1) was observed at left flank (treated with vehicle) of 1/20 animals. A discrete to intense erythema (grade 1 to 3) was noted at right flank (treated with test item) of 5/20 animals, associated with oedema in two of them. A skin dryness was also noted on the right flank (treated with the test item) in 6/20 animals. The cutaneous reactions (grade 1) observed on the right flank treated with the test item of control animals were considered to be attributed to the irritant properties of the test item. Therefore, the discrete erythema observed on the right flank of the test item-treated animals being of similar incidence than in the control group, these reactions were not considered in the evaluation of the results. The cutaneous reactions (grade 2 to 3) observed on the right flank (treated with the test item) of test item-treated animals were considered to be of higher incidence and severity than those recorded at the right flank (treated with the test item) of control animals. As the moderate erythema (grade 2) noted at the 24-hour reading on the right flank (treated with the test item) of one animal became a discrete erythema (grade 1) at the 48-hour reading, it was not considered to be attributed to contact delay hypersensitivity. However, the other findings noted in the test group animals were considered to be attributed to delayed contact hypersensitivity.

 

Under the experimental conditions of this study the substance induced delayed contact hypersensitivity in 3/20 (15%) guinea pigs. However, the test item should not be classified as a skin sensitizer according to the criteria of CLP Regulation.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In a skin sensitisation study performed to OECD TG 406 (Magnusson and Kligman Maximisation Test) the substance induced delayed contact hypersensitivity in 3/20 (15%) guinea pigs. However, the substance should not be classified as a skin sensitizer according to the criteria of CLP Regulation (>=30% positive responses).