Registration Dossier

Administrative data

Description of key information

Skin sensitisation

LLNA (OECD 429): sensitising, EC3 = 3.67% (CIT, 2005)

LLNA (OECD 429): sensitising, EC3 = 6.3% (Basketter et al., 2007)

GPMT (OECD 406): 70% responding at 2% intradermal induction dose (Hoechst AG, 1989)

Human: Resorcinol has elicited allergic skin reactions in patch tests carried out on dermatitis patients.

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
Method: other: OECD TG 429
0.1, 0.5, 1, 5 and 25%
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Nulliparous and non pregnant female CBA/J mice were chosen on the basis of previous studies performed in the laboratory in which CBA/J mice showed the best proliferative response. Females were selected since this sex is recommended by the international guidelines. The Breeder was Janvier, Le Genest-Saint-Isle, France. In the main study 28 females were used (seven groups of 4 animals). On the first day of treatment, the animals were 7 to 12 weeks old and the weight variation of the animals minimal (not exceed 20% of the mean weight). Animals were acclimated at least 5 days before study initiation. Animals were assigned by hand procedure and individually numbered on the tail using an indelible marker.

The animal room conditions will be set as follows: Temp: 22 + 2C; relative humidity: 30 to 70%; light/dark cycle: 12h/12h; ventilation: 12 cycles/hr of filtered, non-recycled air. Animals were housed individually in cages with autoclaved sawdust.

Tap water and A04 pelleted diet were available ad libetum. No containments were known to be present in the diet, drinking water or sawdust (bedding) at levels which may be expected to interfere with or prejudice the outcome of the study.
Vehicle:
dimethylformamide
Concentration:
0.1, 0.5, 1, 5 and 25%
No. of animals per dose:
28 (Five treated groups of four animals received the test item plus controls)
Details on study design:
As equivocal results were obtained in the first experiment (see additional robust study summary) and in order to determine more precisely the EC3 
value, the test item was then tested in a second experiment. 

In the second study:  Five treated groups of four animals receiving the test item Resorcinol (A011) at the chosen concentrations of 0.1, 0.5, 1, 5 and 25%.  According to the solubility assay performed in the CIT/Study No. 26936 AHS, the vehicle used in the study was DMF. During the induction 
phase, the test item, vehicle or reference item was applied over the ears (25 µL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of
resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI). 

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

Proliferation Assay:  The incorporation of 3H-TdR in the vehicle control group was as specified in acceptance criteria, the quantity of cells obtained in each group was satisfactory, the cellularity was correlated with incorporation of 3H-TdR, the cell viability in each group was higher than 70% and the 
threshold positive value of 3 for the SI was exceeded in the positive control group. The study was therefore considered valid.

Intravenous injection of 3H-TdR and sampling of auricular lymph nodes Lymph node cell proliferative responses were measured as described by 
Kimber and Dearman (1991). On day 6 of the experiment, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl
containing 20 µCi of 3H-TdR (specific activity of 25 Ci/mmol) via the tail vein. Approximately 5 hours later, the animals were killed by cervical 
dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group.


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
linear interpolation of the points on the dose response curve immediately above and below the 3-fold threshold (EC3). The equation used for calculation of EC3 was:
EC3 = c + [(3 - d) / (b - d)] x (a - c)
Where a = the lowest concentration giving stimulation > 3; b = the actual stimulation index caused by a; c = the highest concentration failing to produce a stimulation index of 3; and d = the actual stimulation index caused by c.
Positive control results:
Within range.
Parameter:
SI
Value:
1.58
Test group / Remarks:
0.1% test group
Parameter:
SI
Value:
2.87
Test group / Remarks:
0.5% test group
Parameter:
SI
Value:
1.97
Test group / Remarks:
1% test group
Parameter:
SI
Value:
3.51
Test group / Remarks:
5% test group
Parameter:
SI
Value:
5.74
Test group / Remarks:
25% test group
Key result
Parameter:
EC3
Value:
3.67
Remarks on result:
other: The EC3 value was determined by linear interpolation of points on the dose-response curve, immediately above and below the 3-fold threshold. Assuming SI3 is between the concentration 1% and 5%: EC3=1 + [(3-1.97)/(3.51-1.97)] x (5-1) = 3.67

Systemic clinical signs and mortality: Hypoactivity, piloerection and dyspnea were observed on day 3 in 1/4 and 2/4 animals of the treated groups 4 and 5, respectively.  

Local irritation:  No cutaneous reactions and no noteworthy increase in ear thickness were observed in the animals of the treated groups.

Proliferation assay:  The incorporation of 3H-TdR in the vehicle control group was as specified in acceptance criteria, the quantity of cells obtained in each group was satisfactory, the cellularity was correlated with incorporation of 3H-TdR, the cell viability in each group was higher than 70% and the threshold positive value of 3 for the SI was exceeded in the positive control group. The study was therefore considered valid.

A dose-related increase in the stimulation index was noted and the threshold positive value of 3 was exceeded at the concentrations = 5%.

The EC3 value for the test item Resorcinol (A011) calculated on the basis of the results obtained in this experiment was 3.67% assuming SI3 is between the concentration 1% and 5%.


Dose              

# of nodes per group

DPM per group

DPM per node

0.1%

8

514.23

64.28

0.5%

8

933.88

116.74

1%

8

639.94

79.99

5%

8

1142.67

142.83

25%

8

1869.67

233.71

 

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Positive. Resorcinol (AO11) is considered a moderate skin sensitizer under the conditions of this study.

Classification: Skin Sens. 1B, H317
Executive summary:

A mouse local lymph node assay (LLNA) was conducted in accordance with OECD TG 429 (, 2005a; RL=1).  The first experiment resulted in equivocal findings at concentrations of 0, 2.5, 5, 10, 25 and 50%. There were four animals per treatment group including negative and positive controls.  Lymphoproliferative responses observed in negative control groups fell within historical ranges, while significant lymphoproliferation was observed with α-hexylcinnamaldehydr at 25%, thus validating the sensitivity of the test system.  A dose related increase in the Stimulation Index (SI) was not seen. In the second experiment five treated groups of four animals were administered resorcinol at concentrations of 0.1, 0.5, 1.0, 1.5 and 25%.  Clinical signs included hypoactivity, piloerection and dyspnea on day 3 in 1 of 4 animals in the 5% group and 2 of 4 animals in the 25% group.  There was no effect on body weight and no cutaneous reactions; there were no noteworthy increases in ear thickness.  A dose-related increase in the stimulation index was noted and the threshold positive value of 3 was exceeded at concentrations equal to 5%.  Resorcinol was considered positive in the LLNA and a moderate skin sensitizer under the conditions of this study. 

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

Studies in animals

The potential of resorcinol to cause skin sensitization has been investigated in four Local Lymph Node Assays (LLNAs) and one Guinea Pig Maximisation Test (GPMT).

 

A mouse LLNA was conducted in accordance with the principles of GLP and according to OECD TG 429 (CIT, 2005).

To determine the highest non-irritant concentration of resorcinol, a preliminary test was performed. Mice were treated by topical application to the external surface of both ears with test item concentrations of 5, 10, 25 and 50% in dimethylformamide (DMF) once daily each on 3 consecutive days. Measurement of the ear thickness was performed each day before treatment and 24 hours after the last application. Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal achievable concentration (i.e. 50%).

In the first main test, concentrations of 2.5, 5, 10, 25 and 50% resorcinol in DMF were topically applied to four mice per group. During the induction phase, the test item was applied over both ears on days 1, 2 and 3. A vehicle (DMF) control group and a positive control group (25% (v/v) hexyl cinnamic aldehyde in DMF) were maintained under the same environmental conditions and treated in the same manner as the test animals. After 2 days of resting (day 6), the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine. The obtained values were used to calculate stimulation indices (SI). No mortality or any signs of systemic toxicity were observed during the first main test. Lymphoproliferative responses observed in negative control groups fell within historical ranges, while significant lymphoproliferation was observed with hexyl cinnamic aldehyde at 25%, thus validating the sensitivity of the test system. Positive lymphoproliferative responses (SI > 3) were observed at all tested resorcinol concentrations. In the absence of local irritation, these positive responses might be due to skin sensitization reactions. However, no clear dose-related increase was seen in the SI.

In the second main test, five treated groups of four animals were administered resorcinol at concentrations of 0.1, 0.5, 1.0, 1.5 and 25%. Clinical signs included hypoactivity, piloerection and dyspnea on day 3 in 1 of 4 animals in the 5% group and 2 of 4 animals in the 25% group. There was no effect on body weight. No cutaneous reactions or noteworthy increases in ear thickness were observed at any tested concentrations. A dose-related increase in SI was noted and the threshold positive value of 3 was exceeded at concentration ≥ 5%.

 

Table: Skin sensitization potential of resorcinol, LLNA study, second main test (CIT, 2005)

 

No/group

SI

SI > 3

Vehicle

4

1.0

No

0.1% resorcinol

4

1.58

No

0.5% resorcinol

4

2.87

No

1% resorcinol

4

1.97

No

5% resorcinol

4

3.51

Yes

25% resorcinol

4

5.74

Yes

Positive control

4

6.79

Yes

 

According to the study report, the authors concluded that the dose-related increase in SI was not recognized at the concentration of 1%. The SI value at the concentration of 1% was therefore excluded and not used for the calculation of the EC3 value. Assuming SI3 is between the concentration 0.5% and 5%, the authors of this study calculated an EC3 value of 1.4%. However, a clear justification why the SI at the concentration of 1% was not used for the EC3 calculation was not provided in the study report and this calculation appears not to be justified on the basis of the available dose-response.

The EC3 value, as % concentration of test substance required to elicit a SI of 3, was determined by linear interpolation of points on the dose-response curve, immediately above and below the 3-fold threshold. The equation used for calculation of EC3 was: EC3 = c + [(3 - d) / (b - d)] x (a - c); where a = the lowest concentration giving stimulation > 3; b = the actual stimulation index caused by a; c = the highest concentration failing to produce a stimulation index of 3; and d = the actual stimulation index caused by c. According to this method, SI3 is between the concentration of 1% and 5%. Thus, a recalculation on the basis of a more transparent dose-response assumption resulted in an EC3 value for resorcinol of 3.67% (EC3 = 1 + [(3 - 1.97) / (3.51 - 1.97)] x (5 - 1) = 3.67) (Sumitomo Chemical 2012). This calculation is supported by Scientific Committee on Consumer Safety Opinion on Resorcinol (SCCS, 2000). For additional information, if linear regression is carried out by using all SI values, the EC3 is calculated to be even 5.40%, by the formula y = 0.1452x + 2.2161, where y = value and x = concentration (%).

In conclusion, resorcinol induced contact sensitization in this LLNA study. Resorcinol showed an EC3 value of 3.67%. According to the Guidance on the Application of the CLP Criteria (ECHA 2017, Table 3.4.4), the result indicates a moderate skin sensitization potency and suggests Category 1B as the EC3 value is >2%.

 

Three other LLNAs are reported in Basketter et al., 2007. In two studies, that are reported in this article, animals were tested at concentrations of 5, 10, 25% (w/v) and 0.1, 0.25, 0.5, 1.0, 2.5% (w/v) resorcinol. Both studies demonstrated a non-sensitizing potential of resorcinol. However, the authors question these findings as study methods have evolved over time. Within the same article, a newly conducted study following OECD TG 429 is reported. Groups of four CBA/Ca female mice were treated topically to concentrations of 1, 5, 10, 25 and 50% (w/v) resorcinol. The vehicle in this study was a 4:1 ratio of acetone:olive oil (AOO). No discrepancies were noted with the negative and positive controls. A very clear dose response was obtained. SI values of 0.7, 2.2, 5.2, 8.4 and 10.4 were determined with rescorcinol concentrations of 1, 5, 10, 25 and 50% in AOO, respectively. The EC3 value was calculated to be 6.3%, which would allow classification of resorcinol as Category 1B skin sensitiser.

 

The GPMT was conducted according to the principles of GLP and OECD TG 406 (Hoechst AG, 1989). Pirbright White guinea pigs (treatment group 10 animals, control group 5 animals and accompanying group 20 animals) were induced with intradermal injections of 2% resorcinol followed by occlusive, epicutaneous application of 25% resorcinol. Challenge exposure was performed using 25% resorcinol in an occlusive epicutaneous application. There were no clinical signs.

After the first challenge exposure, very slight to clearly circumscribed erythema was observed on the skin of two or three animals in the exposure group 24 and 48 hours, respectively. The skin of the control animals was clear of signs of irritation. After the second challenge exposure, very slight to clearly circumscribed erythema was observed on the skin of 7/10 animals at 24 hours and on the skin of 5/10 animals at 48 hours. Minor swelling was also observed in one animal at 24 hours after bandage removal. The skin of the control animals was clear of signs of irritation. Since 70% of the animals responded at 2% intradermal induction dose, the result indicates a moderate skin sensitization potency and suggests Category 1B according to the Guidance on the Application of the CLP Criteria (ECHA 2017, Table 3.4.4).

Justification for classification or non-classification

Resorcinol shows a moderate skin sensitization potency in mice based on the results of the key LLNA (CIT, 2005). The results meet the criteria for subcategorization, and Category 1B is justified. According to further animal data (Basketter et al., 2007; Hoechst AG, 1989), resorcinol is considered as a moderate skin sensitizer. Resorcinol has elicited allergic skin reactions in patch tests carried out on dermatitis patients.

In conclusion, based on the available data, the proposed classification and labelling for skin sensitization is Category 1B. The corresponding hazard statement is H317: May cause an allergic skin reaction.