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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
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
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
Three experimental groups of five female CBA/J mice were treated with test item concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)).
Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Identification: Resorcinol diacetate
Appearance: Brown liquid
Purity/Composition: 98.52% (GC)
Test item storage: At room temperature
Chemical name (IUPAC), 1,3-Diacetoxybenzene
synonym or trade name
CAS Number: 108-58-7
Molecular formula: C10H10O4
Molecular weight: 194.18
pH (1% in water, 3.60 – 3.51 (determined by WIL Research Europe)
indicative range)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Test System:
Species. Mouse, CBA/J strain, inbred, SPF-Quality. Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA).
Source: Janvier, Le Genest-Saint-Isle, France
Number of animals: 20 females (nulliparous and non-pregnant), five females per group (main study only).
Age and body weight: Young adult animals (approx. 10 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.
Identification. Tail mark with a marker pen.
Health inspection: At least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.
Reliability check: The results of a reliability test with three concentrations of Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures. For both scientific and animal welfare reasons, no concurrent positive control group was included in the study.
An extensive data base is available with reliability checks performed at half year intervals during at least the past 9 years showing reproducible and consistent positive results.

Animal Husbandry:
Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these
conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material. Paper were supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage enrichment.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: Free access to tap water.

Diet, water, bedding and cage enrichment evaluations for contaminants and/or nutrients were performed according to facility standard procedures. There were no findings that could interfere with the study.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0 (acetone/olive oil (4:1 v/v)), 25%, 50%, or 100%.
No. of animals per dose:
5 animals per dose
Details on study design:
Pre-screen Test
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2) and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two test item concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration that could technically be applied.

The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm).
Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

Main Study
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
4.2
Test group / Remarks:
25% concentration
Key result
Parameter:
SI
Value:
9
Test group / Remarks:
50% concentration
Key result
Parameter:
SI
Value:
12.1
Test group / Remarks:
100% concentration
Key result
Parameter:
other: EC3
Remarks on result:
other: An estimated EC3 value of 21% was calculated.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The results show that the test substance elicits a SI ≥ 3. An estimated EC3 value of 21.0% was calculated.
Executive summary:

Three experimental groups of five female CBA/J mice were treated with test item concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone/Olive oil (4:1 v/v)).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal.

After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No irritation was observed in any of the animals. Scaliness, noted for three animals, was considered not to have a toxicologically significant effect on the activity of the nodes.

The auricular lymph nodes of the animals of the control animals, animals treated at 25% and most animals treated at 50% were considered normal in size. The lymph nodes of the animals treated at

100% and two animals treated at 50% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 100% were 2969, 6377 and 8516 DPM, respectively. The mean DPM/animal value for the vehicle control group was 706 DPM. The SI values calculated for the test item concentrations 25, 50 and 100% were 4.2, 9.0 and 12.1, respectively.

These results show that the test substance elicits a SI ≥ 3. Although, the data do not permit the use of linear interpolation, calculation of EC3 values (the estimated test substance concentration that will give a SI =3) by loglinear extrapolation can provide a reliable estimation of sensitization potency class for use in risk assessment and can avoid the need for repeat animal testing. The data show a clear dose response and a slope ratio of 0.32 was calculated indicating that extrapolation can provide a reliable estimation of the EC3 value according to Ryan et al 2007. An estimated EC3 value of 21.0% was calculated.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact

hypersensitivity.

According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Resorcinol diacetate should be classified as skin

sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.