RD 14156

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 20 May 2016 and 08 June 2016

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

22 July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

EC No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor Lot/Batch no.: 5399561P10
- Expiration date of the lot/batch: 25 January 2020
- Purity test date: 25 January 2016


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc, the Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: Suspended solid floor polypropylene cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: >5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 ”C
- Humidity (%): 30 - 70%
- Air changes (per hr): >15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 20 May 2016 To: 08 June 2016

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

Preliminary Screening: 10% w/w in propylene glycol (maximum attainable concentration
Main Test: 10%, 5% and 2.5% w/w in propylene glycol

No. of animals per dose:

Preliminary screening: 1 mouse
Main test 5 mice /concentration

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Maximum attainable concentration = 10% w/w
- Irritation: scored daily
- Systemic toxicity: not determined
- Ear thickness measurements: Between Days 1 and 3 and Days 1 and 6
- Erythema scores: no

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response: Preliminary test: A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.
Main test: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer".

TREATMENT PREPARATION AND ADMINISTRATION:

Positive control substance(s):

other: Phenylacetaldehyde

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine
statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.

Results and discussion

Positive control results:

The positive control Phenylacetaldehyde (>90%) gave a Stimulation Index of greater than 3 (15.12) when tested at a concentration of 5% v/v in propylene glycol, thus, demonstrating the sensitivity and reliability of the test system.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

> 0.92 - < 1.77

Test group / Remarks:

Overall range from all test groups

Results 2

Key result

Parameter:

SI

Remarks:

5%

Value:

> 0.92 - <= 1.77

Results 3

Key result

Parameter:

SI

Remarks:

10%

Value:

> 0.92 - <= 1.77

Results 4

Key result

Parameter:

SI

Remarks:

2.5%

Value:

> 0.92 - <= 1.77

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group

Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Any other information on results incl. tables

Estimation of the Proliferative Response of Lymph Node Cells - Stimulation Index

Concentration in propylene glycol	Stimulation Index	Result
RD 14156 2.5% w/w	0.92	Negative
RD 14156 5% w/w	1.29	Negative
RD 14156 10% w/w	1.77	Negative
Positive control 5% v/v	15.12	Positive

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

non-sensitizer

Conclusions:

The test item was considered to be a non-sensitizer under the conditions of the test.
The positive control Phenylacetaldehyde (>90%) gave a Stimulation Index of greater than 3 (15.12) when tested at a concentration of 5% v/v in propylene glycol, thus, demonstrating the sensitivity and reliability of the test system.

Executive summary:

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

Following a preliminary screening test in which no clinical signs of toxicity were noted at a maximum attainable concentration of 10% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of the test item as a suspension in propylene glycol at concentrations of 10%, 5% or 2.5% w/w. A further group of five animals was treated with propylene glycol alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, Phenylacetaldehyde (>90%), at a concentration of 5% v/v in propylene glycol.

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.

The test item was considered to be a non-sensitizer under the conditions of the test. The positive control Phenylacetaldehyde (>90%) gave a Stimulation Index of greater than 3 (15.12) when tested at a concentration of 5% v/v in propylene glycol, thus, demonstrating the sensitivity and reliability of the test system.