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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:
2016-02-02 to 2016-03-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was performed for regulatory purpose outside EU

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Procured from approved breeder, National Institute of Nutrition
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation: 311.53 g to 321.24 g
- Housing: individually in a standard polypropylene cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:
Healthy young adult animals for pre-study and main study were acclimatized for a period of five and twelve days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1°C to 22.9°C
- Humidity (%): 46% to 63%
- Air changes (per hr): 12 to 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, semiocclusive
Vehicle:
water
Concentration / amount:
5.0% v/v, 100% for intradermal and topical induction, respectively.
Day(s)/duration:
7
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
on day 22
Adequacy of challenge:
other: 100%
No. of animals per dose:
10 Males - Vehicle Control
20 Males - Test Item
Details on study design:
RANGE FINDING TESTS:

MAIN STUDY
A. INDUCTION EXPOSURE
day1: Intrademal injection:
- Site: 3 pair
- Concentrations:
50:50 volume ratio of FCA in distilled water
5% (v/v) Test item in distilled water
5% (v/v) Test item , emulsified in a 50:50 Volume of FCA and distilled water

day 8: Topical application:
100 % (as such) Test item

B. CHALLENGE EXPOSURE
- Day(s) of challenge: day 22
- Concentrations: 100 % (as such) Test item
- Evaluation (hr after challenge): 24±2 and 48±2 hours
Challenge controls:
Distilled water
Positive control substance(s):
yes
Remarks:
2-Mercaptobenzothiazole in study No.: BIO-TX 1685

Results and discussion

Positive control results:
The positive control 2-Mercaptobenzothiazole showed a mean skin sensitisation rate of 60% at approximately 24 and 48 hours post patch removal in challenge phase.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no skin reaction was observed
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no skin reaction was observed
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no skin reaction was observed
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no skin reaction was observed
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
not measured/tested

Any other information on results incl. tables

No treatment related change in body weight and percent change in body weight with respect to Day 1 was noted in all the groups. All animals showed physiologically normal increase in body weights.

No gross pathological changes were noted in any of the treated animals in all the groups.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the above results of the experiment and under experimental conditions employed, it can be concluded that the test item 6-(Isononanoylamino)hexanoic acid, compound with 2,2′,2″-nitrilotriethanol (1:1) did not show any skin sensitisation or allergic potential in Guinea pigs. Hence, the test item is not classified as a sensitizer according to the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals.
Executive summary:

The test item,6-(isononanoylamino)hexanoic acid, compound with 2,2′,2″-nitrilotriethanol (1:1) obtained from Clariant India Private Limited was evaluated for the skin sensitization potential in Guinea pigs as per theOECD Guideline for Testing of Chemicals, Section 4, Number 406, “Skin Sensitization”.

The study was conducted in two phase viz., pre-study and main study.

Pre-study: For pre-study, a total of 4 Guinea pigs were used, 2 Guinea pigs for intradermal injection and 2 Guinea pigs for topical application. Approximately 24 hours before treatment, the fur from the shoulder region and both flank region was clipped for intradermal injection and topical application, respectively. For intradermal injection, 2.5% and 5% (v/v) in distilled water was administered to one animal, and 7.5% and 10% in distilled water was administered to another animal. Pairs of injection, i.e. 0.1 mL/injection were administered at the shoulder region during intradermal injection. For topical application, test concentrations of 25% and 50% v/v in distilled water was applied topically at left and right flank of one animal and 75 % v/v in distilled water and undiluted test item was applied to the left and right flank of another animal.The occlusive patches were removed after 24 hours of contact period and washed with normal saline swabs and dried with absorbent cotton.

After intradermal injection, thetest itemrevealed no erythema at 24 hours and very slight erythema (barely perceptible) at 48 hours in 5.0% (v/v) test item in distilled water. After removal of patch in topical application, thetest itemdid not reveal erythema or oedema with 100% (as such) test item at 24 and 48 hours observation period. Hence, 5.0% v/v of test item in distilled water and 100% (as such) test item was selected for intradermal and topical induction, respectively for the main study, and 100% (as such) test item was selected for challenge phase for main study.

Main study: The main study comprised of two groups, group G2 (10 males) designated as vehicle control (distilled water for intradermal injection, topical application, and for challenge application distilled water and undiluted test item) and G3 (20 males) designated as treatment group 5.0% v/v test item for intradermal induction, 100% as such (undiluted) test item for topical application, and distilled water and undiluted test item for challenge application. Main study included an intradermal induction on Day 1, topical induction on Day 8 and challenge on Day 22. The designated sites for respective phases of the experiment were clipped closely using an electric hair clipper approximately 24 hours prior to initiation of the treatment.

On Day 1 (induction phase), the animals were administered intradermally with 3 pairs of injection at the shoulder region with 0.1 mL/injection. Post intradermal induction, all animals in groups G2 and G3 were scored for skin reactions approximately at 24 and 48 hours as per the Draize (1959) scoring system. Skin reaction like very slight erythema (barely perceptible) was observed at site 2 of intradermal injection in treated group (G3) approximately at 48 hour observation. However, no skin reaction observed at site 2 of vehicle control group (G2) approximately at 24 and 48 hours observation period.

On Day 7, fur from the area of the intra-dermal injection sites on the shoulder region was clipped in both vehicle control and treatment group animals. On Day 8 (induction phase), the filter paper (2 cm x 4 cm) was saturated with distilled water and 100 % as such (undiluted) test item. The filter paper saturated with distilled water was applied to G2 groups and filter paper saturated with 100% as such test item was applied to G3 groups. The soaked filter paper was topically applied to the previously injected sites to the respective groups. The patch was held in place with non-irritating adhesive tape and further wrapped with crepe bandage by an occlusive dressing. The test patch was held in their position for 48±2 hours. After 48±2 hours of contact period the test patch was removed and the area was cleaned with normal saline swab and dried with absorbent cotton. The skin reactions were observed at 1 and 24 hours of post removal of the test patch. The animals did not reveal any skin reactions in G2 and G3 groups.

On Day 22 (challenge phase), the filter paper (2 cm x 4 cm) was saturated with vehicle control and 100% as such (undiluted) test item. The soaked filter paper was applied to the G2 and G3 groups over the pre-clipped area. The vehicle control and test item was applied on the anterior left and posterior left flank region respectively in G2 and G3 groups respectively. After 24±2 hours of contact period, the test patches were removed and the areas was cleaned with normal saline swabs and dried with absorbent cotton. The skin reactions were observed at 24±2 and 48±2 hours after removal of the test patch and the skin reactions were observed and recorded according to Magnusson and Kligman grading scale. There was no skin reactions observed.

No treatment related changes were noted in body weight and percent change in body weight with respect to Day 1 in all the groups. All animals showed physiologically normal increase in body weights.

All the animals were observed once daily for clinical signs of toxicity and twice daily for mortality during observation period. No clinical signs of toxicity and mortality were noted in any of the animals in all the groups.

Body weights were recorded on Day 1 and at termination. At the end of the observation period all the animals were sacrificed under carbon dioxide anaesthesia

No gross pathological changes were noted in any of the animals in all the groups.

Conclusion

Based on the observed results of the experiment and under experimental conditions employed, it can be concluded that the test item 6-(isononanoylamino)hexanoic acid, compound with 2,2′,2″-nitrilotriethanol (1:1) did not show any skin sensitisation or allergic potential in Guinea pigs. Hence, the test item is not classified as a sensitizer according to the Globally Harmonized System (GHS) of Classification and Labelling of Chemicals.