Registration Dossier

Administrative data

Description of key information

Skin sensitisation (in vitro):

This study is waived as a read-across LLNA study to 6,6'-di-tert-butyl-4,4'-thiodi-m-cresol (TBBC, CAS No. 96-69-5).

Read-across from TBBC - Skin sensitisation (in vivo): Sensitisting (Similar or equivalent to OECD429, LLNA)

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
Read-across
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
[Describe why the read-across can be performed (e.g. common functional group(s), common precursor(s)/breakdown product(s) or common mechanism(s) of action]

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
[Provide here, if relevant, additional information to that included in the Test material section of the source and target records]

3. ANALOGUE APPROACH JUSTIFICATION
[Summarise here based on available experimental data how these results verify that the read-across is justified]

4. DATA MATRIX
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
no
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Sigma Chemical Company (St.Louis, MO)
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic (Hudson, NY)
- Age at study initiation: 8–12 weeks
- Housing: Cages were cleaned and sanitized weekly. The NIOSH Animal Facility is an environmentally controlled barrier facility fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.
- Diet: Modified NIH-31 6% irradiated rodent diet (Harlan Teklad #7913) ad libitum
- Water: Tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 and 26°C
- Humidity (%):25–70%
- Photoperiod (hrs dark / hrs light): light–dark cycles at 12-hr intervals
Vehicle:
other: acetone
Concentration:
Range finding: 0.1, 0.5, 1.0, 5.0, 10.0, 12.5, 25, 50 (% w/v)
LLNA: 0.1, 0.5, 1.0, 5.0 and 10.0% (w/v)
No. of animals per dose:
4 females
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Range finding studies were performed to select the concentration of chemicals to be used for sensitization studies. For these studies mice (three per group) were dosed with acetone vehicle (VH), 12.5%, 25%, and 50% of the test article(s) on the dorsal surface of each ear (25 μl per ear) for three consecutive days.
Animals were allowed to rest for 2 d following the last exposure and then weighed and examined for signs of toxicity including loss of body weight and ruffled fur. At the end of the study, mice were sacrificed by CO2 asphyxiation. The highest soluble concentrations were selected for these studies in an attempt to identify toxicity. For the subsequent studies, maximum concentrations were selected that were soluble in the vehicle and did not cause toxicity.

- Irritation & Ear thickness measurements: Before the first chemical administration, the thickness of the right and left pinnae of each mousewas measured using a modified engineer’s micrometer (Mitutoyo Co., Japan). Mice were exposed to 25 μl of VH or test article for three consecutive days. Ear thickness measurements were taken 24 hr following the final exposure. The mean percentage of ear swelling was calculated based on the following equation: [(mean post-challenge ear thickness – mean pre-challenge ear thickness)/mean pre-challenge thickness] × 100. TBBC was tested at a range of 0.1–10%.


MAIN STUDY
The local lymph node assay (LLNA) was performed following the method described in the ICCVAM Peer Review Panel report (NIEHS, 1999) with minor modifications. Briefly, mice (five per group) were exposed topically with VH, increasing concentrations of test article(s), or positive control (30% HCA) on the dorsal surface of each ear (25μl per ear) for three consecutive days (Table 1). Animals were allowed to rest for 2 d following the last exposure. On Day 6, mice were injected intravenously via the lateral tail vein with 20 μCi [3H]-thymidine (Dupont NEN; specific activity 2 Ci/mmol). Five hours after [3H]-thymidine injection, animals were euthanized via CO2 inhalation, and the left and right cervical draining lymph nodes (DLNs) located at the bifurcation of the jugular vein were excised and pooled for each animal.

Single cell suspensions were made and following overnight incubation in 5% trichloroacetic acid (TCA), samples were counted using a Packard Tri-Carb 2500TR liquid scintillation analyzer. Stimulation indices (SI) were calculated by dividing the mean disintegrations per minute (DPM) per test group by the mean DPMfor the vehicle (VH) control group. EC3 values (concentration of chemical required to induce a 3-fold increase over the VH control)were calculated based on the equation from Basketter and colleagues (Dearman et al., 1999).

TBBC was tested at 0.1, 0.5, 1.0, 5.0 and 10.0% (w/v) in the LLNA assay.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis was performed using Graph Pad Prism version 3.0 (San Diego, CA). All data were analyzed by a one-way analysis of variance (ANOVA). In the ANOVA, when significant differences were detected (p= 0.05), Dunnett’s test was used to compare treatment groups with the appropriate control
group. Statistical significance is designated by *p < 0.05 and **p < 0.01.
Positive control results:
The positive control gave the appropriate response (data not shown).
Parameter:
SI
Value:
1.13
Test group / Remarks:
0.1% (w/v)
Parameter:
SI
Value:
6.87
Test group / Remarks:
0.5% (w/v)
Parameter:
SI
Value:
14.28
Test group / Remarks:
1% (w/v)
Parameter:
SI
Value:
20.19
Test group / Remarks:
5% (w/v)
Parameter:
SI
Value:
15.3
Test group / Remarks:
10% (w/v)
Parameter:
EC3
Value:
0.23
Variability:
%
Test group / Remarks:
TBBC
Cellular proliferation data / Observations:
TBBC was evaluated for toxicity using a 6-day range finding study. At the concentrations tested, no overt toxicity or body weight loss was reported for TBBC (data not shown).

TBBC did not induce a significant irritancy response as demonstrated by the absence of an increase in ear swelling 24 hr post-challenge (data not shown).

The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was greater than 0.23 % (w/v).

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
In an LLNA in female BALB/c mice, TBBC is a skin sensitiser with an EC3 value of 0.23% (wv).
Executive summary:

In a dermal sensitization study (Myers et al., 2007) with 0.1, 0.5, 1.0, 5.0 and 10.0% (w/v) TBBC in 100% acetone), young adult BALB/c mice (5 females) were tested in the Local Lymph Node Assay. The reliability of the test system was confirmed by concurrent testing of the positive control 30% α- hexylcinnamaldehyde in acetone.

The positive control, Hexylcinnamaldehyde, gave the appropriate response. Treatment with TBBC at 0.1, 0.5, 1.0, 5.0 and 10.0% (w/v) in 100% acetone resulted in resulted in stimulation indices of 1.13, 6.87, 14.28, 20.19 and 15.3 respectively. The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was 0.23 % (w/v).

In this study, TBBC is a dermal sensitizer.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin sensitisation (in vitro):

This study is waived as a read-across LLNA study to 6,6'-di-tert-butyl-4,4'-thiodi-m-cresol (TBBC, CAS No. 96-69-5) is available.

Skin sensitisation (in vivo):

In a dermal sensitization study (Myers et al., 2007) with 0.1, 0.5, 1.0, 5.0 and 10.0% (w/v) in 100% acetone), young adult BALB/c mice (5 females) were tested in the Local Lymph Node Assay. The reliability of the test system was confirmed by concurrent testing of the positive control 30% α- hexylcinnamaldehyde in acetone.

The positive control, Hexylcinnamaldehyde, gave the appropriate response. Treatment with TBBC at 0.1, 0.5, 1.0, 5.0 and 10.0% (w/v) in 100% acetone resulted in resulted in stimulation indices of 1.13, 6.87, 14.28, 20.19 and 15.3 respectively. Consequently, the test substance is designated as likely to be a sensitiser under the conditions of the test.  The estimated concentration giving rise to a 3 fold increase in lymphocyte proliferation (EC3) was greater than 0.23 % (w/v).

In this study, TBBC is a dermal sensitizer. Based on this study, UV1084 is also predicted to be a dermal sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available information in the dossier, the substance UV-1084 (CAS No. 14516-71-3) needs to be classified for skin sensitisation (Category 1A) when the criteria outlined in Annex I of 1272/2008/EC and Annex I of 286/2011/EC are applied, based on the results of the read-across study from TBBC (CAS No., 96-69-5).