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Description of key information

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:
other: Well conducted standard guideline OECD study according to GLP
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Test System
Species : Guinea pigs
Strain : Albino , NIH (Dunkin Hartley)
Source : Toxicology
Department of Safety Assessment Advinus Therapeutics Private Limited Bangalore 560 058, INDIA
Rationale for selection of Guinea pigs : Skin reactions in the guinea pigs are classically used for determining the potential of the test substance to induce delayed contact hypersensitivity

No. of groups / animals: 2 groups

Control Group - 10 (5 Males + 5 Females)
Treatment Group - 20 (10 Males + 10 Females)
Note: The results of Historical positive control data of G6951 is included

Age : Young adults (10 – 12 weeks)
Body weight : < 500g body weight at intra-dermal injection
Identification : Guinea pig accession number. Identification of individual animals is by cage card and body markings using turmeric solution / crystal violet solution.

Acclimatization : After physical examination for good health and suitability for the experiment, the animals were acclimatized for 5 days before start of the treatment. Females were nulliparous and non-pregnant. At the end of acclimatization period, the animals procured for the study were weighed and the body weights are arranged in ascending order. The animals were distributed to each of the study groups with in the ratio of 1:2 for G1 and G2 groups respectively. Example: The first animal in the order was allotted to G1 group and the second and third animal is allotted to G2 group and continued similarly with G1 and G2 groups to attain the required number of animals for the respective groups.

Conditions
Animals were housed under standard laboratory conditions, air conditioned with adequate fresh air supply (12-15 air changes/hour). Environment: temperature 20 - 22°C relative humidity 56-65%, with a 12 hour light and 12 hour dark cycle. The maximum and minimum temperature and relative humidity in the experimental room was recorded once daily. The relative humidity in the experimental room was calculated from dry and
wet bulb temperature recordings.

Housing
Guinea pigs were housed individually in standard polysulfone cages (Size: approximately L 425 x B 266 x H 175 mm), with stainless steel top grill having facilities for pelletted food and drinking water; bedding: steam sterilized clean paddy husk was used and changed along with the cage twice a week.

Diet ad libitum
Guinea pig feed manufactured by Pranav agro Industries Ltd, Sangli, Maharashtra, India was provided ad libitum to the animals. The procured feed was subjected to analysis of composition.

Water ad libitum
Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cumpurifier manufactured by Eureka Forbes Ltd., Mumbai - 400 001, India, was provided. The water bottles were replenished once daily and the water bottles changed once a week.
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
3% v/v of the test substance in Distilled water was selected for the intradermal induction of the main study.
75% v/v of the test substance in distilled water was selected for the topical induction
50% v/v of the test substance in distilled water was selected for the challenge application of the main study.
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
3% v/v of the test substance in Distilled water was selected for the intradermal induction of the main study.
75% v/v of the test substance in distilled water was selected for the topical induction
50% v/v of the test substance in distilled water was selected for the challenge application of the main study.
No. of animals per dose:
Control - 10 (5m, 5f)
Test - 20 (10m, 10f)
Details on study design:
TEST SUBSTANCE PREPARATION
Induction (intra-dermal injection)

Control group:
Solution-1: 20 mL of 50% v/v CFA mixture (10 mL of CFA + 10 mL of distilled water) with 20 mL of distilled water was mixed as a 1:1 mixture and vortexed.
Solution-2: Distilled water.
Solution-3: 10 mL of solution 1 with 10 mL of solution 2 was mixed as a 1:1 mixture and vortexed.

Treatment group:
Solution-4: 3% v/v of test substance in distilled water : 0.6 mL of test substance was made up to 20 mL in distilled water and vortexed.
Solution-5: 0.6 mL of test substance made up to 10 mL with distilled water (i.e., 6% v/v) and added to 10 mL of solution 1 (10 mL of 1 : 1 mixture of solution-1 and solution-2) to achieve the final concentration of 3 % v/v and vortexed.

Boosting
75% v/v – of the test substance in distilled water: 11.25 mL of the test substance was dissolved and made up to 15 mL with disilled water.

Challenge
50% v/v – of the test substance in distilled water: 7.5 mL of the test substance was dissolved and made up to 15 mL with distilled water.

The main test comprised of 3 phases, viz.,
1) INDUCTION: Intradermal injection
2) INDUCTION (BOOSTING): Topical application
3) CHALLENGE: Topical application

1. INDUCTION
Intradermal injection
The hair of all the animals was removed from the shoulder region (at least an area of 2 x 4 cm) using an electric clipper (Aesculap ® Germany), approximately 24 hours before administration.
Day 1: The animals were injected using sterile 1 mL tuberculin syringe fitted with 26 x ½" gauge needle at the shoulder region with 0.1 mL each of 3 pairs of intradermal injections, so that one injection of each pair is on either side of the midline. Injections 1 and 2 were given near the head region 1 cm apart and injection 3 towards caudal part of the body.

2. INDUCTION (BOOSTING)
TOPICAL APPLICATION
On day 7, the intra dermal injection site (approximately 2 x 4 cm area) was closely clipped. Approximately 24 hours after clipping, ie., on day 8, the test item at the concentration of 75% v/v in distilled water was added drop by drop to the filter paper (2 x 4 cm) until it was fully loaded and applied on to the skin (site of intradermal injection) and covered with cotton gauze (size: 3 x 4 cm - 6 ply). This was held in place for 48 hours by adhesive tape (for occlusion) and crepe bandage wound around torso over which a clean cotton cloth (many tailed bandage) was tied to anchor the crepe bandage.
Control group: Handled similar to treatment group animals but without test substance.

3. CHALLENGE
TOPICAL APPLICATION
The treatment group and control group animals were challenged on the 22nd day of the test (the day of intradermal injections to animals was reckoned as day '1' of the test), by topical application of the test item at the concentration of 50% v/v in distilled water to the control and treatment group animals.
i. One day before the challenge application on day 21, the hair on both the flanks of the animal approximately 80 sq. cm (8 x 10 cm) was closely clipped.
ii. A fully loaded filter paper strip (size; 2 x 4 cm) with the test item at the concentration of 75% v/v in distilled water was applied to the prepared (clipped) area of skin of the left flank. The patches applied on the skin was covered by a cotton gauze (size: 3 x 4 cm – 6 ply). Similarly, a fully loaded filter paper strip with distilled water was applied on to the right flank. The applications were held in place with the help of adhesive tape and crepe bandage wound around torso over which a clean cotton cloth (many tailed bandage) was tied to anchor the crepe bandage. These challenge patches were kept in contact with the skin for a period of 24 hours. On day 23, the patches were removed and the area of skin where the patch was applied was cleaned with mild soap solution and dried.
iii. 24 hours after the removal of patches (viz 48 hours after challenge application), the skin was examined for skin reaction which was scored using Buehler method (1975).
iv. Skin was observed again at 48 hours after the removal of the patch or on day 25 (72 hours after the challenge application) in order to confirm the previous day’s observations. All the observations were recorded.
9.5 OBSERVATIONS
9.5.1 TOXIC SIGNS AND PRE-TERMINAL DEATHS
Test animals were observed for toxic signs and pre-terminal deaths at approximately 2, 4 and 6 hours post administration on treatment days, twice daily on evaluation days and once daily on other days till the termination of the experiment.
9.5.2 BODY WEIGHTS
The body weights were recorded at the start of the treatment (pre-administration), at weekly intervals thereafter and at termination.
9.5.3 SKIN SCORING
i. Observations for skin reactions were made at 24 and 48 hours after the Induction (intradermal administration). Scoring of the administration sites were recorded according to the scheme originally developed by Buehler and Griffin (1975).
ii. Induction patch sites were observed at 1 and 24 hours post removal of the test patch (topical application). Scoring of the administration sites were recorded according to the scoring scheme originally developed by Buehler and Griffin (1975).
iii. Challenge sites were observed at 24 and 48 hours, post removal of the test patch (topical application), at both the right and left flank. Scoring of the administration sites were recorded according to the scoring scheme originally developed by Buehler and Griffin (1975).

EVALUATION OF SKIN REACTIONS
BUEHLER SENSITIZATION SCORING SYSTEM
Skin Reaction Value
0 = No reaction
0.5 = Very faint reaction, usually confluent
1 = Faint erythema, usually confluent
2 = Moderate erythema
3 = Strong erythema with or without edema

9.5.4 NECROPSY AND HISTOPATHOLOGY
All the surviving animals were subjected to gross necropsy at the end of the observation period. Guinea pigs were euthanized via isoflurane anaesthesia. External surface of the body, all orifices, tissues and organs of the thoracic and abdominal cavities of all animals were examined. All necropsy observations were recorded.
Positive control substance(s):
no
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.

Toxic Signs and Pre-Terminal Deaths:

There were no toxic signs and pre-terminal deaths

Body Weights:

All the Guinea pigs gained weight through the observation period

Skin Reactions

a. Control group (G1):

Intradermal induction: At 24 and 48 hours (post administration) observation period, there were no skin reactions in any of the treated sites.

Topical boosting: There were no skin reactions at 1 and 24 hours post removal of the test patch.

Challenge: There were no skin reactions at 24 and 48 hours post removal of the test patch.

b. Historical positive control data [(2-Mercaptobenzothiazole) of Study No. G6951]:

Challenge application: Six out of 10 guinea pigs had score of 1 (faint erythema, usually confluent) at 24 hours and five out of 10 guinea pigs had score of 1 (faint erythema, usually confluent) at 48 hours post removal of the test patch.

c. Treatment (G2):

Intradermal induction : At 24 and 48 hours (post administration) observation period, there were no skin reactions in any of the treated sites.

Topical boosting: At 1 and 24 hours post removal of test patch, very faint changes (score value of 0.5) at the site of treatment was observed in 17 out of 20 animals.

Challenge: There were no skin reactions at 24 and 48 hours post removal of the test patch.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The study showed that the test item XU-12323.00 Experimental Amino Alcohol pH adjusted to 9.5 with concentrated Hydrochloric acid is a “Non-sensitiser” to Guinea pigs under the stated experimental conditions.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In spite of the corrosivity of the test material it was possible to conduct the mouse LLNA using test material concentrations up to 80% in propylene glycol. This is likely due to the small volume of material used in this study compared to that used in the irritation/corrosivity assay (25 microlitres compared to 500 microlitres) and the buffering capacity of the vehicle used.

In the mouse local lymph node assay performed using octanolamine the concentration associated with a 3 fold increase over control in the stimulation index (the EC3 value) was determined to be 21%. This is consistent with weak dermal sensitisation potential as described by an Expert ECETOC panel (Technical Report No. 87, 2003).

Octanolamine does not posess any structural alerts for skin sensitising potential and is not a 'reactive' substance therefore it is considered possible that the result of the LLNA is actually a false positive. There have been indications that substances with strong irritation potential can produce 'false' positive results when tested in the mouse LLNA (sodium decyl sulphate for example). In order to clarify this result, a Guinea pig maximisation study, a protein reactivity assay and ARE cytotoxicity assay were conducted.

In the guinea pig assay there were no positive reactions in any of the animals at either the 24 or the 48 hour challenge. The doses tested were equivalent to those in the local lymph node assay. Considering the complete lack of response in any animals in this study it appears that the LLNA assay and the GPMT have a significant disagreement. In order to clarify which assay was most likely predicting the sensitising potential for humans, additional mechanistic assays were conducted.

In the first assay the peptide reactivity of 3 -amino-4 -octanol was assessed. This assay demonstrated that the test material was not capable of binding directly to proteins and indicates that it is highly unlikely to be a Hapten (i.e. a sensitiser). This confirmed the initial analysis using DEREK and TOPKAT that did not find any stuctural alerts.

In the second assay the ability of the substance to intitiate the Antioxidant Response Element pathway in cultured human keratinocytes was assessed. This pathway has been demonstrated to be associated with intracellular responses to sensitisers. The assay is currently under development as a potential in vitro replacement assay for sensitising assessment. The keratinocyte cell line also has endogenous metabolic potential and so the assay is also capable of identifying pro-haptens. In this assay, the test material did not trigger the Antioxidant response element pathway at any concentration tested. As such it was considered highly unlikely that this material would be a human sensitiser.

Based on the available data, the weight of evidence indicates that 3 -amino-4 -octanol is unlikely to be a relevant sensitiser in humans. The difference in the mouse and guinea pig assay could be due to confounding by irritancy in the mouse assay. The same issue has been identified with a number of other substances including surfactant and surfactant-like molecules, where positive LLNA data do not agree with negative guinea pig and human data.


Migrated from Short description of key information:
OECD Guideline mouse LLNA study., GPMT, Peptide Reactivity, ARE assay

Justification for selection of skin sensitisation endpoint:
High quality, reliability 1 OECD guideline study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

No data.


Migrated from Short description of key information:
No data available

Justification for classification or non-classification

Based on the weight of evidence, this substance is not considered to be a skin sensitiser.