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Diss Factsheets

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:
09 Feb 2015 - 10 Feb 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD and EC test guidelines and in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
The theory behind the local lymph node assay (LLNA) is that a sensitizing substance induces proliferation of lymphocytes in the lymph nodes draining the site of test substance exposure. The lymphocyte proliferation is proportional to the dose and potency of the sensitizing substance and is
quantified by measuring the incorporation of radiolabelled Thymidine. Assessment of sensitization potential is made by comparing the mean
proliferation in each test group to the mean proliferation in the vehicle control group, with the ratio between treated and control groups termed
the Stimulation Index (SI). A test substance is regarded as a sensitizer if the SI is 3 or more in any of the concentrations tested.
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Automatically generated during migration to IUCLID 6, no data available
IUPAC Name:
Automatically generated during migration to IUCLID 6, no data available
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Unjecol 84AN
- Physical state: Liquid
- Analytical purity: 98%
- Lot/batch No.: 5253
- Expiration date of the lot/batch: 30 October 2015
- Storage condition of test material: Room Temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
The mice were in the weight range 15.0 to 20.7 g and approximately eight to twelve weeks of age prior to dosing on Day 1. They were acclimatised to the experimental environment for at least 6 days prior to the start of the study.

Each animal was assigned an alpha-numeric code and identified uniquely within the study by tail marking. Each cage label was colour-coded and was identified uniquely with the study number, test substance concentration and animal mark(s).

Animals were housed inside a barriered rodent facility The facility was designed and operated to minimise the entry of external biological and chemical agents and to minimise the transference of such agents between rooms.

The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature and relative humidity controls were set to maintain the range of 19 to 23C and 40 to 70% respectively. Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study. Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours.

Periodic checks were made on the number of air changes in the animal rooms. Temperature and humidity were monitored daily and records are archived with the departmental raw data. Alarms were activated if there was any failure of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available to be automatically brought into operation should the public supply fail.

The animals were allowed free access to a standard rodent diet. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent. Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.

Each batch of diet was analysed routinely by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinised and approved before any batch of diet was released for use. The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. Certificates of analysis were received routinely from the water supplier.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, open
Vehicle:
olive oil
Concentration / amount:
50% v/v in 4:1 v/v acetone:olive oil
Challenge
Concentration / amount:
50% v/v in 4:1 v/v acetone:olive oil
No. of animals per dose:
4 females per dose, 24 females in total.
Details on study design:
Preliminary investigation
Two female mice (per concentration) received a daily application of 25 µL of appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette.

Main phase
Groups of four mice were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette. Further groups of four mice received the vehicle alone or the positive control substance in the same manner.

Administration of 3H-methyl Thymidine
In the main phase of the study, five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline containing 3H-methyl Thymidinea (3HTdR: 80 µCi/mL) giving a nominal 20 µCi to each mouse. The injection into the tail vein was carried out using a plastic syringe and needle after the mouse had been heated in a warming chamber.
Positive control substance(s):
yes
Remarks:
25% v/v hexyl cinnamic aldehyde

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10, 25 % v/v of test substance
No. of animals per dose:
Four female mice per group.
Details on study design:
The theory behind the local lymph node assay (LLNA) is that a sensitizing substance induces proliferation of lymphocytes in the lymph nodes draining the site of test substance exposure. The lymphocyte proliferation is proportional to the dose and potency of the sensitizing substance and is quantified by measuring the incorporation of radiolabelled Thymidine. Assessment of sensitization potential is made by comparing the mean proliferation in each test group to the mean proliferation in the vehicle control group, with the ratio between treated and control groups termed the Stimulation Index (SI). A test substance is regarded as a sensitizer if the SI is 3 or more in any of the concentrations tested.

Results and discussion

Positive control results:
For Preliminary and Main study phases

There were no deaths and no signs of ill health or toxicity were observed during this study. Greasy fur on the head was noted following each dosing occasion, this was related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance

No signs of dermal irritation were seen on the ear during the study.

Ear measurements on Day 6, when compared with Day 1 indicated there was no evidence of an effect of treatment on ear thickness in mice dosed at 25% v/v.
Ear thickness means of greater than 25% were seen in both mice dosed at 50%v/v along with. eschar/scab formation on the ears seen in one mouse (No. B2).

There was no indication of an effect of treatment on body weight gain.
A loss in body weight over the study period was noted for one control group mouse (No. B5) and one mouse (No. B15) dosed at 10% v/v, however, a small loss in body weight is not uncommon in young laboratory mice and is not considered to be an effect of treatment.

The SI for the positive control substance hexyl cinnamic aldehyde was 16.0, which demonstrates the validity of this study.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SI obtained for 5, 10 and 25% v/v were 1.0, 2.7 and 4.7 respectively which indicates that UNJECOL 85AN showed the potential to induce skin sensitization.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Acetone:olive oil (4:1) (vehicle control): 4392.15 dpm 5% v/v: 4201.15 dpm 10% v/v: 12001.35 dpm 25% v/v: 20822.95 dpm Hexyl cinnamic aldehyde (positive control) 25% v/v: 70442.95 dpm

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information
Conclusions:
UNJECOL 85AN is regarded as a potential skin sensitizer. The EC3 value was calculated to be 12.3% v/v.