RD 14153

Endpoint summary

Description of key information

The test item is considered to be a non-sensitizer under the conditions of a local lymph node assay.

No information is available for respiratory sensitization, however, the substance is a waxy solid with a low vapour pressure and therefore respiratory sensitization is considered unlikely.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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 02 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sponsor Batch/Lot No. 5399661C10
- Expiration date of the lot/batch: 19 January 2020
- Purity test date: 19 January 2016

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity:
- Specific activity:
- Locations of the label:
- Expiration date of radiochemical substance:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:

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 wks
- 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: None

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 ”C
- Humidity (%): 30 - 70%
- Air changes (per hr): >15/h
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 20 May 2016 To: 01 June 2016

Vehicle:

propylene glycol

Concentration:

Preliminary screening = 10% w/w in vehicle
Main Test = 10%, 5% or 2.5% w/w in vehicle

No. of animals per dose:

Preliminary screening = one mouse at 10% w/w
Main Test = 5/dose level

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: 10% w/w = maximum attainable concentration in vehicle
- Irritation:
- Systemic toxicity:
- Ear thickness measurements: mean thickness >25% indicated excessive irritation
- Erythema scores:

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:
- Criteria used to consider a positive response: 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:
Groups of five mice were treated with the test item at concentrations of 10%, 5% or 2.5% w/w in propylene glycol. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.
The positive control animals were similarly treated to the test animals except that 25 μL of the positive control item, Phenylacetaldehyde (>90%), at a concentration of 5% v/v in propylene glycol was applied to the dorsal surface of each ear.

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.

Positive control results:

Positive control dosed at 5% w/w in propylene glycol gave a positive response with a Stimulation Index of 9.67

Key result

Parameter:

SI

Remarks:

2.5% w/w in propylene glycol

Value:

1.22

Test group / Remarks:

negative

Key result

Parameter:

SI

Remarks:

5% w/w in propylene glycol

Value:

0.99

Test group / Remarks:

negative

Key result

Parameter:

SI

Remarks:

10% w/w in propylene glycol

Value:

1.07

Test group / Remarks:

negative

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA See Table 1

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

EC3 CALCULATION

CLINICAL OBSERVATIONS: There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS 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.

Table 1: Estimation of the Proliferative Response of Lymph Node Cells (Cellular Proliferation Data)

Treatment Group	Concentration in Vehicle	Stimulation Index	Result
Test Item	2.5% w/w	1.22	Negative
Test Item	5.0% w/w	0.99	Negative
Test Item	10% w/w	1.07	Negative
Positive control	5% w/w	9.67	Positive

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 (9.67) when tested at a concentration of 25%

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 are as in Table 1 above.

Conclusion

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 (9.67) when tested at a concentration of 25% v/v in propylene glycol, thus, demonstrating the sensitivity and reliability of the test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. 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.

Stimulation index was <3 at all three test concentrations (SI 1.22 at 2.5%, SI 0.99 at 5% and SI 1.07 at 10%)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance has been tested and found not to be a skin sensitzer with a Stimulation index of <3.

In accordance with the classifciation criteria described in CLP Regulation 1272/2008 the substance is not classified for skin sensitization.

No information is available for respiratory sensitization however, the substance is a waxy solid with a low vapour pressure and therefore respiratory sensitization is considered unlikely.