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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 31/01/2012 to 12/03/2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented GLP study performed according to a method similar to OECD TG 406 without significant deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
Suitable non-LLNA data was already available. Therefore the generation of new LLNA data is not necessary.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: clear colorless liquid
Details on test material:
- Name of test material (as cited in study report): n-Methylmorpholine Oxide 50% (NMMO)
- Substance type: Organic
- Physical state: Clear colourless liquid
- Analytical purity: 50%
- Composition of test material, percentage of components: 14 ppb Nitrosomorpholine, 50.0 wt% N-methylmorpholine oxide, 5 ppm Hydrogen peroxide, 6.9 ppm Nitrate, 0.02 wt% NMM, 0.1% Total other amines
- Purity test date: 09/01/2012
- Lot/batch No.: F30-T151-291211
- Expiration date of the lot/batch: Not declared
- Stability under test conditions: Stability data on the bulk test material was not provided by the Study Director
- Storage condition of test material: Room temperature (21-29°C)
- Other:

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elm Hill Breeding Laboratories
- Age at study initiation: 4 weeks (dose range); 5 weeks at start of dosing (Day 1) of the main study
- Weight at study initiation: 307 - 335 grams (dose range); 319-448 grams at the outset (Day 1) of the main study
- Housing: Animals were individually housed upon receipt and upon assignment to study in compliance with USDA Guidelines. The room in which the animals were kept was documented in the study records. No other species were kept in the same room.
- Diet (e.g. ad libitum): All animals had access to Harlan Teklad Guinea Pig Diet (certified) ad libitum.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Study animals were acclimated to their housing for a minimum of 5 and 7 days respectively for the dose range and main assay prior to their first day of dosing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 26 °C
- Humidity (%): 13 to 68%
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
The dose chosen was 100% (as received - i.e. 50% NMMO).
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
The dose chosen was 100% (as received - i.e. 50% NMMO).
No. of animals per dose:
Total number:
4 Primary irritation (Dose Range)
20 Test Article Group (10 male/10 female)
10 Vehicle Control Group (5 male/5 female)
6 Positive Control Group (3 male/3 female)
Details on study design:
RANGE FINDING TESTS:
Each side of four naive animals were clipped free of hair the day prior to dose. Four concentrations of the test article (10ù, 25%, 50% or 100%) were dosed on four separate sites. The location of each of the four applications of the test article differed in each of the four animals to compensate for any site-to-site variations. For grading of the response, the procedure described below for primary challenge was used, except that only 24-hour grades were obtained. The concentration chosen for induction was generally one that produced mild irritation. The highest concentration that was used for challenge was one that induced no scores > 1 and scores did not exceed two [±] in the group of four animals.

MAIN STUDY
A. INDUCTION EXPOSURE
The test article or vehicle or positive control was applied to a 25 mm Hill Top Chamber patch. The animal was placed in a restrainer and the patch applied to the clipped surface as quickly as possible after the test article preparation has been applied to the patch. The patch was occluded with a rubber dental dam by pulling it tightly over the animal and fastening it to the bottom of the restrainer with binder clips. The restrainer was adjusted to minimize movement of the animal during the exposure period. Both edges of the dental dam were under the front and back adjustable braces of the restrainer.

Six (± 30 min) hours (4.5 to 5 hours-for the first induction) later, the dental dam and patch were removed and the animal was placed in its cage. The test article preparations were removed by gently wiping with water and gauze before returning the animals to their cages. The treated sites were examined after each dosing day and scored at 20-30 and 44-54 hours. This dosing and scoring procedure was done within 3 weeks for a total of three 6-hour exposures to the test article.

B. CHALLENGE EXPOSURE
Fourteen days after the last induction exposure, the animals were challenged in the same manner but the patches were applied to freshly clipped skin sites that have not been previously. Since the vehicle used for induction was not water (saline) and was the same for both induction and challenge, the test animals were also challenged with vehicle.
Twenty ( ± 2) hours after removal of the challenge patch, all animals were depilated with Nair Lotion Hair Remover. The depilatory was placed on the test sites and surrounding areas and left on for six minutes. The depliatory was then thoroughly washed off with water and animals patted dry and returned to their cages. The inside of each cage was wiped to remove any depilatory that might have contaminated the cage. Approximately four hours after depilation, test sites were graded such that the grading was done 24 hours after removal of the challenge patch (24-hour grade). The grading was repeated 24 hours later (48-hour grade).

OTHER:
In-life observations and measurements:
1. Mortality:
Frequency: once daily
2. Clinical observations:
Frequency: once daily
3. Dermal observations
Frequence: The treated sites were examined after each dosing day and scored at 24 and 48 hours after dosing. Following the challenge, approximately four hours after depilation, test sites were graded such that the grading was done 23 hours after removal of the challenge patch (24-hour grade). The grading was repeated 24 hours later (48-hour grade).
Dermal irritation was scored according to following grades:
No reaction: 0
Slightly patchy erythema: ±
Slight or confluent or moderate patchy erythema: 1
Moderate erythema: 2
Severe erythema with/without edema: 3
Terminal procedures and anatomic pathology:
1. Termination:
a) Scheduled sacrifice:
All animals were euthanized by CO2 inhalation/asphyxiation following final skin/dermal grading.
2. Gross necropsy:
No gross necropsy was performed at experimental completion. Carcasses were disposed of accordingly.
Challenge controls:
The vehicle control group was challenged with the test article and vehicle.
Positive control substance(s):
yes
Remarks:
DNCB (@ 0.3% in 80% EtOH for induction; @ 0.2% in Acetone for challenge)

Results and discussion

Positive control results:
The positive control group, induced and challenged with DNCB, exhibited the anticipated positive responses at challenge, indicating that the methods employed in this study were valid.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
NMMO at 100%, saline
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of systemic toxicity
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: NMMO at 100%, saline. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No signs of systemic toxicity.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
NMMO at 100%, saline
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No signs of systemic toxicity
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: NMMO at 100%, saline. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No signs of systemic toxicity.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
NMMO at 100%, saline
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No signs of systemic toxicity
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: NMMO at 100%, saline. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No signs of systemic toxicity.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
NMMO at 100%, saline
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No signs of systemic toxicity
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: NMMO at 100%, saline. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No signs of systemic toxicity.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.2% DNCB
No. with + reactions:
6
Total no. in group:
6
Clinical observations:
No signs of systemic toxicity
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.2% DNCB. No with. + reactions: 6.0. Total no. in groups: 6.0. Clinical observations: No signs of systemic toxicity.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.2% DNCB
No. with + reactions:
4
Total no. in group:
6
Clinical observations:
No signs of systemic toxicity
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.2% DNCB. No with. + reactions: 4.0. Total no. in groups: 6.0. Clinical observations: No signs of systemic toxicity.

Any other information on results incl. tables

Clinical observations:

There were no signs of systemic toxicity and all animals gained weight during the main study.

Challenge phase:

All dermal irritation scores at 24- and 48 -hour observations were zero for the vehicle control animals challenged with the test article and vehicle (saline).

All dermal irritation scores at the 24 -and 48 -hour observations were zero for the test article group animals challenged with the test article and the vehicle (saline).

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study, induction with NMMO (n-Methylmorpholine Oxide 50%) as received did not elicit a delayed contact hypersensitivity response in guinea pigs when challenged as received.