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Description of key information

Under the conditions of a Bühler test, induction with NMMO (n-Methylmorpholine Oxide 50%) as received did not elicit a delayed contact hypersensitivity response in guinea pigs when challenged with a 50% aqueous solution.

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 31/01/2012 to 12/03/2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
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.
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
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
100 % aqueous solution containing 50 % NMMO
Day(s)/duration:
6h
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
100 % aqueous solution containing 50% NMMO
Day(s)/duration:
6 h
Adequacy of challenge:
other: highest technically applicable concentration used
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)
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.
Key result
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:
no indication of skin sensitisation
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:
no indication of skin sensitisation
Key result
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:
no indication of skin sensitisation
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:
no indication of skin sensitisation
Key result
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:
positive indication of skin sensitisation
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:
positive indication of skin sensitisation

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).

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

Vasquez RE (2012) investigated the sensitising properties of NMMO 50% in a Buehler test with male and female Hartley guinea pigs. The study examined the skin sensitising effect on guinea pigs: 4 for primary irritation (dose range), 20 for test article group (10 male/10 female), 10 for vehicle control group (5 male/5 female) and 6 for positive control group (3 male/3 female).

Positive and vehicle controls were included in the study. Animals were induced and challenged with the commercial NMMO (as received, i.e. 50%). 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.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the criteria of the CLP Regulation, NMMO does not have to be classified as sensitising to the skin.