Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 to 23 November 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Principles of method if other than guideline:
Assessment of lymph node proliferation using BrdU incorporation assessed using ELISA method
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 4,4-biphenol
- Physical state:powder
- Analytical purity:99.9%
- Purity test date: 9 September 2011
- Lot/batch No.: 071111370
- Expiration date of the lot/batch: 11 July 2016
- Storage condition of test material: Ambient room temperature
- Other: description off white to white cream coloured powder

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/JN (CBA/J)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CBA/JN (CBA/J) mice obtained from Charles River Italia S.p.A. Calco, Italy
- Age at study initiation:7-8 weeks old (21-25g) at time of ordering. circa 8-9 weeks at study initiation
- Weight at study initiation: 18.0-20.4g
- Housing:The animals were housed in a limited access rodent facility. The animals will be housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm. Nesting material will be provided inside suitable bedding bags; nesting material will be changed at least twice a week.
- Diet (e.g. ad libitum): ad libitum , laboratory rodent diet (4RF18, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI))
- Water (e.g. ad libitum):ad libitum, Records of analyses of water and diet are kept on file at RTC.

- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):Animal room controls will be set to maintain temperature and relative humidity at 22°C + 2°C.
- Humidity (%):55% + 15%
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 8 November 2012 To: 23 November 2012

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Local lymph node assay - Preliminary phase
Four concentrations (25, 10, 5 and 2.5% w/w in acetone:olive oil 4:1 (v/v) were selected to be used in the preliminary phase, to identify a non toxic and minimally irritant concentration in order to avoid false positive results. No signs of toxicity (clinical signs or toxicologically relevant body weight losses) were observed at any concentration tested. According to the results of the irritation screening, the highest concentration tested (25% w/w) was judged not irritant and was selected as starting dose level for the main assay. A 50% formulation was prepared but was found not to be administerable.

Main assay
In the main assay, the test item was topically administered at the concentrations of 25, 10 and 5% w/w, in acetone:olive oil 4:1 (v/v).
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: yes
- Irritation: yes (assessed by dermal reaction, ear thickness measurements and ear biopsy weights)
- Lymph node proliferation response: not assessed in preliminary phase

At least five concentrations (2.5; 5; 10; 25 and 50% in acetone:olive oil) of the test item in the selected vehicle and the vehicle alone were tested in 1-2 animals for each test concentration. The 50% formulation could not be administered suitably.
Healthy stock animals with no observable skin lesions were selected and allocated to the preliminary phase (on Day 1). Each mouse was treated for three consecutive days by topical application to the dorsal surface of each ear with 25 µl of the appropriate concentration of test item formulation or with the vehicle alone (Days 1, 2 and 3). The application volume was spread over the entire dorsal surface of the ear. Mortality, morbidity, clinical signs of reaction to application and bodyweights were checked regularly from Day 1 to termination on Day 6. Dermal reactions at site of application were recorded and ear thickness measurements were recorded on Days 1,3 and 6. At necropsy ear biopsies were obtained for tissue weights.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA BrdU-ELISA
- Criteria used to consider a positive response: SI of greater than or equal to 1.6

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance was administered at the concentrations that were systemically tolerated and judged not to cause excessive reaction at the treatment site in the preliminary phase. Dose concentrations of 5, 10 and 25% v/v in acetone:olive oil were selected based on the preliminary test results. Concentrations were considered irritant if :
erythema grading (score) was ≥ 3 and/or
ear thickness was ≥ 25 % and/or
ear punch weight was ≥ 25 %
with reference to the pre-dose scores and/or to negative control group, at any day of measurement, during the preliminary phase.

Groups of four female mice were treated with vehicle or the positive control (HCA) or one of three test substance concentrations 5, 10 and 25% v/v in acetone:olive oil.
Each group of mice was treated for three consecutive days by topical application to the dorsal surface of each ear with 25 µl of the appropriate concentration of either test or positive control items or with the vehicle alone (Days 1, 2 and 3). The application volume was spread over the entire dorsal surface of the ear. On Day 5, a solution of 5-bromo-2-deoxyuridine (BrdU), at a concentration of 10 mg/mL in physiological saline was administered to each animal by intraperitoneal injection.
The dose volume was 0.5 mL/animal.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
statistical analysis of the stimulation indices used to determine positive response or significant dose related increase in lymph node proliferation

Results and discussion

Positive control results:
SI value for HCA was greater than the threshold value of 2.0, validating the methods used in the assay and confirming acceptable positive control response.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.98
Test group / Remarks:
Low tested concentration
Key result
Parameter:
SI
Value:
1.32
Test group / Remarks:
Intermediate tested concentration
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
High tested concentration
Key result
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: BrdU labelling per group: control mean - 0.099 group 2: 5% - 0.196 group 3: 10% - 0.130 group 4: 25% - 0.197 group 5 positive control - 0.490

Any other information on results incl. tables

No mortality and no clinical signs were recorded for the mice treated at any of the three dose levels, nor for the controls. Changes in body weight observed during the study were within the expected range for this strain and age of animals.

Increases in cell proliferation were observed in the low and high dose groups. The stimulation index (SI) was calculated to be 1.98 (low dose group) and 2.00 (high dose group). The animals of the mid-dose group showed an increase in cell proliferation in comparison with the controls - the stimulation index was 1.32 for this group. Although this increase was not greater than the value of SI that indicate the potential of the test item to induce sensitisation (≥ 1.6), it is sufficient to have at least one of the groups showing an increase ≥ 1.6, to consider the test item as a potential sensitiser. In addition, the response was statistically significant at the low and high dose levels when compared to controls. Therefore, the results obtained in this study indicate that the test item may elicit a sensitisation response in mice following dermal exposure. In the group treated with the positive control item, a SI of 4.97 was calculated. As it was greater than 2, the test system was regarded as valid.

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The SI (Stimulation Index) was calculated as 1.98, 1.32 and 2.00 for the low, medium and high exposure groups respectively. The response was statistically significant (p< 0.05) for the low and high dose groups. A positive response is indicated where the SI > 1.6 and consequently classification will be required for biphenyl-4,4'-diol, as 'H317: may cause an allergic skin reaction' in accordance with Regulation 1272/2008. The classification is category 1 with the signal word "Warning".
Executive summary:

The potential of the test item, 4,4 Biphenol, to cause skin sensitisation reactions following topical application to the skin of CBA/JN (CBA/J) mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline for testing of chemicals no. 442b. Preliminary phase Four concentrations (25, 10, 5 and 2.5% w/w in acetone:olive oil 4:1 (v/v) were selected to be used in the preliminary phase, to identify a non toxic and minimally irritant concentration in order to avoid false positive results. No signs of toxicity (clinical signs or toxicologically relevant body weight losses) were observed at any concentration tested. According to the results of the irritation screening, the highest concentration tested (25% w/w) was judged not irritant and was selected as starting dose level for the main assay. Main assay In the main assay, the test item was topically administered at the concentrations of 25, 10 and 5% w/w, in acetone:olive oil 4:1 (v/v). No mortality and no clinical signs were recorded in animals treated at all dose levels. Changes in body weight observed during the study were within the expected range for this strain and age of animals. Increases in cell proliferation were observed in low and high dose levels. The calculated stimulation index (SI) was equal to 1.98 (low dose group) and 2.00 (high dose group). The animals of the mid-dose group showed an increase in the stimulation index (1.32). Although this increase was not greater than the value of SI that indicate the potential of the test item to induce sensitisation (≥ 1.6), it is sufficient to have at least one of the groups an increase ≥ 1.6, to consider the test item as a potential sensitiser. In addition, the response was statistically significant at the low and high dose levels when compared to controls. Therefore, the results obtained in this study indicate that the test item may elicit a sensitisation response in mice following dermal exposure. In the group treated with the positive control item, a SI of 4.97 was calculated. As it was greater than 2, the test system was regarded as valid. European Directives concerning the classification, packaging and labelling of dangerous substances (Council Regulation (EC) No. 1272/2008 and subsequent revisions) would indicate the following: Classification : Category 1; Signal word : Warning; Hazard statement : H317: May cause an allergic skin reaction.