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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04/02/2016-23/02/2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to the OECD and GLP guidelines without significant deviations.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name of the substance: Alpha-methylstyrene
EC number: 202-705-0
CAS number: 98-83-9
Appearance: clear colorless liquid
Batch number: 011080116F
Storage conditions: room temperature in the dark
Purity: 99.783 % (m/m)

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc, Horst, The Netherlands
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15 ACH
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary test: 25µL of undiluted test item
Main test: 100%, 50%, 25% v/v in acetone/olive 4:1
No. of animals per dose:
5 mice/dose
Details on study design:
PRELIMINARY SCREENING TEST:
Using available information regarding the systemic toxicity/irritancy potential of the test item, a preliminary test was performed using one mouse.
- Treatment: daily application of 25 µL of undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- Observation: mouse observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
- Reporting: Local skin irritation was scored daily
- Clinical observation: Yes (the body weight was recorded on Day 1 (prior the dosing) and on Day 6.
- Ears thickness: the thickness of each ear was measured using Mitutoyo 547-300S gauge, pre-dose on Day 1, post -dose on Day 3 and on Day 6. Mean ear thickness changes were calculated between time periods Days 1 and Days 3 and Days 1 and 6. 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 STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Individual method
- Criteria used to consider a positive response: the test item will be regarded as positive if at least one concentration of the test substance results in a three-fold or greater increase in HTdR incorporation compared to control values.

TREATMENT PREPARATION AND ADMINISTRATION:
The mice were treated by daily application of 25µL of the appropriate concentration of the test substance to the dorsal surface of each ear for 3 consecutive days (Day 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.
Five days following the first topical application the test item or control (vehicle) (Day 6) all mice were injected with 250 µL of phosphate buffered saline (PBS) containing 3H-methyl thymidine giving a total of 20µCi to each mouse.

OBSERVATIONS:
- Clinical observations: animals were observed twice per day on Days 1, 2 and 3 and on daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Body weight: recorded on day 1 & day 6
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data were processed to give group mean values for disintegrations per minute and standard deviations were appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data were first assessed for suitability by analysis of normality and homogeneity of variance. If the assumption that the data are both normally distributed and has homogeneity of variances were met, then parametric one way analysis of variance (ANOVA) and Dunnett's multiple comparison procedure were used to determine statistical significance. If the assumption were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.

Results and discussion

Positive control results:
At a concentration of 25%(v/v) the positive control shows a Stimulation Index of 6.08. Therefore, the Hexyl cinnamic aldehyde, tech., 85% is considered to be a sensitizer under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Vehicle = n.a. ; Test substance 25%: 2.35 ; Test substance 50%: 3.13 ; Test substance 100%: 4.50
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Vehicle = 661.04 (+/-262.06) mean dpm/animal (standard deviations) Test substance 25%: 1550.37 (+/- 63017) mean dpm/animal (standard deviations) Test substance 50%: 2071.57 (+/-478.40) mean dpm/animal (standard deviations) Test substance 100%: 2971.66 (+/-288.36) mean dpm/animal (standard deviations)

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test substance was considered to be sensitizer under the conditions of the test.
Executive summary:

The test has been performed according to the OECD 429 and GLP guideline without significant deviations.

The 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 concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three group, each five animals, were treated with 50µL (25µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentration of 50% or 25% v/v.

A control group of five animals were treated with acetone/olive oil 4:1 alone.

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 follows:

Concentration (%v/v) in acetone/olive oil 4:1 Stimulation Index Results
Vehicle na na
25 2.35 Negative
50 3.13 Positive
100 4.50 Positive

The concentration of the test item expected to cause a 3 fold increase in radioactive incorporation (EC3 value) was calculated to be 46%.

In conclusion:

Test item is to be considered as a sensitizer under the conditions of the test.