Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Currently no LLNA study is available for assessment. The Guinea Pig Maximization Test (GPMT) has been carried out as an animal test to predict human sensitization for over a decade and is recommended by international test guidelines such as OECD.
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Hartley guinea pigs were received from Elm Hill Breeding Labs, Inc., Chelmsford, MA. Animals were individually housed upon arrival in compliance with USDA guidelines. Water and feed were provided ad libitum. For the definitive study, the animals were acclimated for at least 7 days prior to dosing. Guinea pigs weighed 312 437 g and were at least 35 days old at the start of dosing. The temperature and humidity were maintained at 15-25°C and 10-97%, respectively. Room lights were on a 12-hour light/dark cycle.
Route:
intradermal and epicutaneous
Vehicle:
other: Mineral oil for intradermal dose and petrolatum for epicutaneous dose
Concentration / amount:
1% TMBPA in mineral oil for intradermal induction;
25% TMBPA in petrolatum for topical induction and challenge doses.
Route:
epicutaneous, occlusive
Vehicle:
other: Mineral oil for intradermal dose and petrolatum for epicutaneous dose
Concentration / amount:
1% TMBPA in mineral oil for intradermal induction;
25% TMBPA in petrolatum for topical induction and challenge doses.
No. of animals per dose:
4 male/4 females for the preliminary irritation test
10 males/10 females Test group (25% TMBPA)
5 males/5 females Vehicle control group
3 males/3 females Positive control group
Details on study design:
Preliminary irritation range-finding tests were conducted to determine the intradermal and topical dose concentrations to be used in the main sensitization study. For the intradermal test 2 guinea pigs per sex were injected intradermally with 4 concentrations of TMBPA in mineral oil (1.0, 2.5, 4.0 and 5.0%). The 1% and 2.5% concentrations were difficult to dose and the 4.0 and 5.0% concentrations were nearly impossible to dose even with larger gauge needles. The injection sites were graded 24-hours after dosing. For the topical range-finding test 2 guinea pigs per sex were dosed topically with 4 concentrations of TMBPA in petrolatum (1, 5, 10 and 25%). The location of each concentration differed for each of the 4 animals to compensate for any site-to-site variation. The procedures described for the main study topical induction period was used except that the wrapping was removed at 24 hours and 5 minutes after dosing. Approximately 20 hours after unwrapping the sites were depilated and approximately 4 hours after depilation the sites were examined for skin reactions and scored.

Based on the results of the preliminary dose-ranging finding tests, the intradermal challenge dose concentration selection was 1% TMBA and 25% TMBPA was the concentration selected for the topical induction and challenge doses.

The hair from an area over the shoulders was removed using clippers 24 to 48 hours prior to the intradermal induction doses. On Day 1 of the main study, each guinea pig received intradermal injections (0.1 mL each) at six sites between the shoulder based on the following table where sites 1 and 2 were the more anterior sites and FCA (1:1) = Freund’s Complete Adjuvant (50:50 dilution with distilled water):

Vehicle Control Group:
Sites 1 & 2 = FCA (1:1)
Sites 3 & 4 = Mineral oil
Sites 5 & 6 = Mineral oil in FCA (1:1)

Test Substance Group:
Sites 1 & 2 = FCA (1:1)
Sites 3 & 4 = 1% TMBPA in mineral oil
Sites 5 & 6 = 1% TMBPA in FCA (1:1)

Positive Control Group:
Sites 1 & 2 = FCA (1:1)
Sites 3 & 4 = 0.1% DNCB in mineral oil
Sites 5 & 6 = 0.1% DNCB in FCA (1:1)

Topical Induction Procedures: On Day 7 the test sites were treated with 10% sodium lauryl sulfate (in petrolatum) since the test substance was not irritating. On Day 8 (one week after the intradermal injections), the test substance (25% in petrolatum) was spread over a 2 x 4 cm filter paper, saturated (0.3 mL) and applied to the injection site area of the test substance group animals and occluded using Blenderm® tape. The dressing was secured with additional bandaging. After a minimum of 48 hours of exposure the dressings were removed. The vehicle and positive control animals were exposed to the vehicle and positive control materials, respectively, using the same procedure.
Challenge controls:
Challenge Period: Fifteen days after the topical induction (Day 22), the test substance group animals, and vehicle and positive control group animals were challenged with occluded patches for 24 hours on the clipped (approximately 30 hours prior) test sites on the right and left flanks. For the test substance and vehicle control groups, a 2 x 2 cm filter paper was saturated with 0.2 mL of the test substance at 25% in petrolatum and applied to one flank and a 2 x 2 cm filter paper, saturated with 0.2 mL of petrolatum, was applied to the opposite flank. The positive control animals were similarly treated with the positive control article (0.05% in petrolatum) on one flank and the vehicle (petrolatum) on the opposite flank. The same occlusive technique used during the induction phase was employed. The wrappings were removed approximately 24 hours later and the sites were wiped clean with gauze and water. The challenge sites of all animals were depilated (using Nair® Lotion Hair Remover) 21±1 hour after unwrapping and 3 to 5 hours later the sites were graded for elicited skin reactions (24-hour scoring period). The sites were scored again approximately 24 (±2) hours later (48-hour scoring period). The sites were scored using the following grading system:

0 = No visible change
1 = Discrete or patchy erythema
2 = Moderate and confluent erythema
3 = Intense erythema and swelling

All animals were also observed daily for clinical signs of toxicity or mortality. Body weights were obtained prior to dose administration and at final (48-hour) skin scoring at the challenge. After the final challenge scoring all animals were euthanized by CO2 inhalation and discarded without further examination.
Positive control substance(s):
yes
Remarks:
Dinitrochlorobenzene (DNCB)
Positive control results:
A sensitization response was observed in all positive control animals confirming the validity of the test method.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.05% in petrolatum
No. with + reactions:
6
Total no. in group:
6
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.05% in petrolatum. No with. + reactions: 6.0. Total no. in groups: 6.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.05% in petrolatum
No. with + reactions:
6
Total no. in group:
6
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.05% in petrolatum. No with. + reactions: 6.0. Total no. in groups: 6.0.

Preliminary Irritation Trial: Intradermal challenge: 24-hours after injection with 1% TMBPA, 2 of 4 animals exhibited a score of 1 and the other 2 sites did not elicit a response. Three of 4 injection sites had a score of 2, 24 hours after injection with 2.5% TMBPA and scores of 2 were observed at all 4 injection sites 24 hours after injections of 4% or 5% TMBPA. No signs of irritation were observed in any animal after topical application of TMBPA at 25%, 5% or 1% in petrolatum. At 10% TMBPA in petrolatum, 2 of 4 injection sites exhibited scores of 1, the other two sites elicited no response.

Main study:

All animals gained weight during the study and no mortality or clinical signs of toxicity were observed. No test or vehicle control animals showed signs of erythema or edema at the 24 and 48 hour challenge observation periods. The positive control group animals exhibited erythema grades 2 and 3 at both the 24 and 48 hour challenge observation periods. The severity of the responses post-challenge were calculated as follows:

 

Negative Control

Test

Positive Control

 

24 hour

48 hour

24 hour

48 hour

24 hour

48 hour

Severity*

0/10 = 0

0/10 = 0

0/20 = 0

0/20 = 0

17/6 = 2.8

15/6 = 2.5

*Severity = The sum of the test reaction grades divided by the number of animals tested.

 

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, TMBPA was not considered to be a skin sensitizer since none of the test animals exhibited erythema or edema at the challenge exposure following an induction phase. No reactions were observed in the negative control group and 100% reactivity was observed in the positive control group at challenge indicating the validity of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Under the conditions of this study, none of the test animals exhibited erythema or edema at the challenge exposure following an induction phase. No reactions were observed in the negative control group and 100% reactivity was observed in the positive control group at challenge indicating the validity of the test. Therefore, TMBPA was not considered to be a skin sensitizer.


Migrated from Short description of key information:
A skin sensitisation test on guinea pigs was performed with TMBPA under GLP/OECD Guideline 406 to assess skin sensitisation. Under the conditions of this study, TMBPA was not considered a skin sensitizer.

Justification for classification or non-classification

Based on the negative results in a skin sensitisation study, the test substance does not need to be classified according to Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.