Registration Dossier

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
When the GPMT was performed, the LLNA did not yet exist as an OECD guideline.
Specific details on test material used for the study:
- Name of test material (as cited in study report): TK 10048
- Analytical purity: 100.1 %
- Lot/batch No.: EN 325964.12
- Expiration date of the lot/batch: June, 1996
- Storage condition of test material: Room temperature
- Physical state: solid
Species:
guinea pig
Strain:
other: Pirbright White strain (Tif: DHP)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Ciba-Geigy Ltd, Animal Production, 4332 Stein, Switzerland
- Age at study initiation: adult
- Weight at study initiation: 352 - 409 g
- Housing: individually in Macrolon cages (Type 3)
- Diet: NAFAG No. 845 (Gossau AG); ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Route:
intradermal and epicutaneous
Vehicle:
other: intradermal injections: Oleum arachidis and saline; epidermal applications: vaseline
Concentration / amount:
Intradermal injection: 0.1 mL of a 5% mixture of test substance in Oleum arachidis
Epidermal: 0.4 g of a 30% mixture of the test substance in vaseline

Route:
epicutaneous, occlusive
Vehicle:
other: intradermal injections: Oleum arachidis and saline; epidermal applications: vaseline
Concentration / amount:
Epidermal: 0.2 g of a 20% mixture of the test substance in vaseline
No. of animals per dose:
10 animals/sex/dose
Details on study design:
PRE-TEST:
- The concentration for the intradermal injections was selected on account of the solubility of the test article in standard vehicles and its local and systemic tolerability. The following concentration of test article have been prepared for intradermal injection: 1 and 5% in oleum arachidis. Since 5% was well tolerated, this concentration was used for intradermal induction.
- The concentrations for epidermal applications were selected on account of the primary irritation potential of the test article. The following concentrations have been examined on separate animals for the determination for the maximum subirritant concentration: 1, 5, 10 and 30% in vaseline. The tested concentrations did not induce erythema reactions. 30% were chosen for epidermal induction and 20% for epidermal challenge application.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous), one week between intradermal und epidermal exposure
- Exposure period: 48 h for epicutaneous
- Test groups: 1). FCA/saline 1:1 mixture, 2). test substance in Oleum arachidis, 3). test substance in FCA/saline mixture
- Control group: same as the test group with the exception that the test substance was ommited
- Site: neck
- Frequency of applications: single

B. CHALLENGE EXPOSURE
- No. of exposures: single
- Day(s) of challenge: 29 (2 weeks after epidermal induction)
- Exposure period: 24 h
- Test groups: Test substance in vaseline and vaseline alone
- Control group: Vaseline and test substance on separate flanks
- Site: flank
- Evaluation (hr after end of challenge exposures): 24 and 48


Positive control substance(s):
yes
Remarks:
The sensitivity of the strain is checked every 6 months with at least one of the following known sensitiser: 2,4-dinitrochlorobenzene, paraphenylene diamine or potassium dichromate.
Positive control results:
Positive control test performed using 1-chlor-2,4-dinitrobenzol between June 17, 1991 and July 18, 1991. In the test group, test article treated animals showed 10/10 animals with positive responses at 24 and 48 hours after challenge. Vehicle control animals showed 0/10 animals with positive responses at 24 and 48 hours after challenge. In the negative control group, there were 0/10 animals with positive responses at 24 and 48 hours after challenge in both the vehicle control and test article treated animals.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20%
No. with + reactions:
1
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20%
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10

Mean body weights (g)

Control

Test group

Control

Test group

Males

Females

Day 0

390

386

381

376

End of study

596

605

577

553

Interpretation of results:
GHS criteria not met
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
other: OECD Draft Guideline: Acute Dermal Photoirritation Dose-Response Test, February 1995
Qualifier:
according to guideline
Guideline:
other: CTFA Safety Testing Guidelines: The Cosmetic, Toiletry and Fragrance Association, Inc. Washington, DC 20036; Guidelines for Evaluating Photodermatitis, 1991
Principles of method if other than guideline:
This test was designed to determine if the test substance, when applied topically to guinea pigs, will induce a photoallergic response.
GLP compliance:
not specified
Type of study:
other: Photoallergy test
Justification for non-LLNA method:
When the test was performed, the LLNA did not yet exist as a guideline test.
Specific details on test material used for the study:
- Lot/batch No.: 005271D0AA
Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Ace Animals, Boyertown, PA, USA
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: 300 - 450 g
- Housing: Stainless steel cages
- Diet: Lab Diet Certified Guinea Pig Diet #5026; ad libitum
- Water: ad libitum
- Acclimation period: 22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 29.4
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route:
epicutaneous, open
Vehicle:
petrolatum
Concentration / amount:
Induction: 100%
Route:
epicutaneous, open
Vehicle:
petrolatum
Concentration / amount:
Challenge: 100, 50 or 20%
No. of animals per dose:
20 test group animals
10 negative control group animals
10 positive control group animals (5 control group animals were used for the positive control experiments)
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
A.1. EPICUTANEOUS
- Site: nuchal skin
- Frequency of FCA/saline intradermal injections: once, on day 1
- Frequency of epicutaneous applications: day 1 (after FCA injection) then on days 3, 6, 8 and 10, making a total of 5 exposures
- Test groups: 4 intradermal injections of 0.1 mL FCA/saline (1:1) followed by epicutaneous test substance (100%; 0.1 g)
- Negative control group: 4 intradermal injections of 0.1 mL FCA/saline (1:1). No epicutaneous inductions were performed
- Positive control: 4 intradermal injections of 0.1 mL FCA/saline (1:1) followed by epicutaneous control substance (7.5% in ethanol; 0.1 mL)
- Exposure period: 2 weeks
- Duration of exposure: not applicable

A.2. IRRADIATION
- Frequency of applications: after epicutaneous treatments on days 1, 3, 6, 8 and 10
- Type of light: UV-B and UV-A
- Dosage: two minimal erythemal dosage of UV-B and 10 J/cm² UV-A

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: approximately day 22 (3 weeks after beginning of induction)
- Exposure period: up to 72 hours
- Test groups: 100, 50 or 20% in petrolatum (0.025 mL/cm²)
- Negative control group: petrolatum and ethanol
- Positive control: 7.5, 5 or 2.5% in ethanol (0.025 mL/cm²)
- Site: dorsal
- Evaluation (hr after start of challenge): 24, 48, 72 hours

B.1. CHALLENGE IRRADIATION
- Dosage: 10 J/cm² UV-A inadiation.
- Comment: left side (irradiated), right side (no radiation)

OTHER: A multi-port solar simulator from the Solar Light Company was utilized. The output in UV-B includes the 280-320 nm (UV-B) range and extends to 360 nm. The output in UV-A includes the 320-400 nm range. Each irradiated site was circular and approximately one cm in diameter.

Positive control substance(s):
yes
Remarks:
3,3',4',5-tetrachlorosalicylanilide
Positive control results:
No. of positive animals/number of animals tested
Test group:
- 7.5%: 9/10 (24h), 10/10 (48h), 10/10 (72h)
- 5.0%: 8/10 (24h), 10/10 (48h), 10/10 (72h)
- 2.5%: 5/10 (24h), 10/10 (48h), 8/10 (72h)
- 0% (ethanol): 1/10 (24h), 2/10 (48h), 1/10 (72h)

Control group
- 7.5%: 1/5 (24h), 0/5 (48h), 0/5 (72h)
- 5.0%: 0/5 (24h), 0/5 (48h), 0/5 (72h)
- 2.5%: 0/5 (24h), 0/5 (48h), 0/5 (72h)
- 0% (ethanol): 0/5 (24h), 0/5 (48h), 0/5 (72h)
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50 and 100%
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50 and 100%
No. with + reactions:
0
Total no. in group:
20
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
50 and 100%
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
20%
No. with + reactions:
4
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
20%
No. with + reactions:
7
Total no. in group:
20
Reading:
other: 3rd Reading
Hours after challenge:
72
Group:
test chemical
Dose level:
20
No. with + reactions:
5
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0%
No. with + reactions:
1
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0%
No. with + reactions:
2
Total no. in group:
20
Reading:
other: 3rd Reading
Hours after challenge:
72
Group:
test chemical
Dose level:
0%
No. with + reactions:
2
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50 and 100%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 and 100%
No. with + reactions:
0
Total no. in group:
10
Reading:
other: 3rd Reading
Hours after challenge:
72
Group:
negative control
Dose level:
50 and 100%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
20%
No. with + reactions:
1
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
20%
No. with + reactions:
3
Total no. in group:
10
Reading:
other: 3rd Reading
Hours after challenge:
72
Group:
negative control
Dose level:
20%
No. with + reactions:
1
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
2
Total no. in group:
10
Reading:
other: 3rd Reading
Hours after challenge:
72
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10

No indication of sensitizing or photosensitizing potential was observed.

Results of non-irradiated skin (right side)

(No. of positive animals/number of animals tested)

Test group:

- 100%: 0/20 (24h), 0/20 (48h), 0/20 (72h)

- 50%: 0/20 (24h), 1/20 (48h), 1/20 (72h)

- 20%: 1/20 (24h), 4/20 (48h), 3/20 (72h)

- 0% (vaseline): 1/20 (24h), 2/20 (48h), 2/20 (72h)

Control group

- 100%: 0/10 (24h), 0/10 (48h), 0/10 (72h)

- 50%: 0/10 (24h), 0/10 (48h), 0/10 (72h)

- 20%: 1/10 (24h), 2/10 (48h), 0/10 (72h)

- 0% (vaseline): 0/10 (24h), 0/10 (48h), 0/10 (72h)

Interpretation of results:
GHS criteria not met
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
(before topical treatment, animals were injected with the test substance in FCA, sacrifice one day after the last topical treatment, ³HTdR incoporation was performed in vitro instead of injecting the test animals)
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-(2'-hydroxy-3'-tert-butyl-5'-methylphenyl)-5-chlorobenzotriazole; Tinuvin 326
- Source: Japan Ciba Geigy Inc., Japan
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan SLC Inc., Shizuoka
- Age at study initiation: 7 - 10 weeks
- Sex: female
Vehicle:
other: DMSO (Intradermal injections), acetone/olive (4/1) (topical treatments)
Concentration:
Injection (intradermal): 0.02, 0.2%
Challenge (topical): 2%
No. of animals per dose:
3
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The result was considered positive in this test, if exposure to the test chemical resulted in an incorporation of ³HTdR at least three-fold greater than that observed in vehicle treated control mice.

TREATMENT PREPARATION AND ADMINISTRATION:
At least mice per group were injected intradermally with 50 µL of the test chemical in FCA (Freund's complete adjuvant) into the shaved abdomen. 5 days after the injection, mice were exposed topically (ear) to 25 µL of various concentrations of the test chemical in vehicle or vehicle alone on both ears daily for 3 consecutive days. The day after the final exposure, mice were sacrificed and the draining auricular lymph nodes were excised. Lymph nodes were pooled per each experimental group and a single cell suspension of LNC was prepared. Cells were cultured (5 culture wells per experimantal group) with 0.5 µCi ³HTdR for 24 h and ³HTdR incorporation was determined by liquid scintillation counting.
Parameter:
SI
Remarks on result:
other: no data.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: no data Results were present as graphs and not in tabulated form. Treatment with the test substance did not affect proliferative response of the auricular draining lymph nodes.
Interpretation of results:
GHS criteria not met
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
4 - 5 mice per group were intradermally injected the test substance in FCA into the shaved abdomen and after 5 days treated topically with the test substance on 3 consecutive days. 7 days after the final exposure, ear thickness was measured using an engineer's micrometer and the mice were challenged with the test substance in vehicle or vehicle alone. Ear thickness was measured again 24 h after and the percentage increase was determined. Seven days after the primary challenge, each group of mice was rechallenged with 2% of the test substance in acetone/olive oil (4:1) and elicitation reactions were measured.
GLP compliance:
not specified
Type of study:
mouse ear swelling test
Justification for non-LLNA method:
When the test was performed, the LLNA did not exist as a guideline study.
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-(2'-hydroxy-3'-tert-butyl-5'-methylphenyl)-5-chlorobenzotriazole;
Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan SLC Inc., Shizuoka, Japan
- Age at study initiation: 7 - 10 weeks
Route:
intradermal
Vehicle:
DMSO
Concentration / amount:
0.02 and 0.2%
Route:
other: topical
Vehicle:
acetone/olive oil (4:l v/v)
Concentration / amount:
2%
No. of animals per dose:
5
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: MEST
- Criteria used to consider a positive response: The result was considered positive if the ear thickness following challenge was at least 20% greater than that before challenge.

Challenge with 2% of the test substance did not produce a positive reaction in mice. (Results were presented as plotted diagrams). Figures were not given.

Interpretation of results:
GHS criteria not met
Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In the key study, the potential of the test article (no data on purity) to cause "delayed contact hypersensitivity" after dermal contact was appraised in Pirbright White (Tif: DHP) Guinea Pigs in a Guinea Pig Maximization Test. Intradermal inductions were performed with 5% of the test substance in Oleaum arachidis. Topical induction (occlusive; 48 hours) was carried out with 30% of the test substance in Vaseline. Challenge exposures were performed (epicutaneous occlusive; 24 hours) with 30% of the test substance in Vaseline. The control and test groups consisted of 10 and 20 animals, respectively. Evaluation of the skin reactions were performed 24 and 48 hours after the end of challenge exposures according to the Draize method. The sensitivity and reliability of the experimental technique was ascertained in the lab every 6 months using 2, 4-dinitrochlorobenzene, paraphenylene diamine or potassium dichromate

After challenge, 1 out of 20 guinea pigs exhibited evidence of "delayed contact hypersensitivity" at the 24 hour observation. At the 48 hour check, no animal gave positive results. All animals of the negative control group did not show evidence of "delayed contact hypersentisation" (0/20) (Ciba Geigy Ltd. 1991).

This study is acceptable as it satisfies the requirement for OECD Guideline 406 for Skin Sensitisation data. In addition, the study was performed under GLP.

In a modified version of the Mouse Lymphoma Assay, an increase in the stimulation index above 3 was not detected when 3 female Balb/c mice per group were injected intradermally with a mixture of the test substance (0.02 and 0.2%) in DMSO and FCA, 5 days prior to topical auricular applications (3 consecutive days) of 2% of the test substance in acetone/olive oil (4:1)). Negative control animals were treated with the vehicles (Ikaraschi et al. 1993).

Likewise, the test substance did not cause a positive reaction in a Mouse Ear Swelling Test (MEST) performed by Ikaraschi et al. (1993). In the test, 5 mice per group were injected intradermally with a mixture of the test substance (0.02 and 0.2%) and FCA. Five days later, topical auricular applications with 2% of the test substance in acetone/olive oil (4.:1)) were performed on 3 consecutive days. Seven days later, ear thickness was measured using an engineer's micrometer and then the mice were challenged. Ear thickness was measured again 24 h after challenge and the percentage increase was determined. A rechallenge with 2% of the test substance in acetone/olive oil (4:1) was performed 7 days after challenge. Negative controls animals were treated with the vehicles. An increase in ear thickness above 20% in comparison to controls indicated a positive response.

In a photosensitization assay, twenty 4 – 6 weeks old male Hartley guinea pigs were first given injections of a FCA/saline adjuvant and then five successive inductional epicutaneous (open) treatments with the undiluted test substance; immediately after FCA injections, on day 3, 6, 8 and 10. After each treatment, the application site was irradiated with 2 MED (minimal erythemal dosage) UV-B and 10 J/cm2UV-A light. Twelve days later, challenge exposures with the undiluted test substance as well as with 50 and 20% of the test substance in Vaseline were performed. Challenged sites were irradiated with 10 J/cm2 UV-A light. Treated, but non-irradiated areas served as controls. In addition, ten animals treated with the adjuvant only served as negative controls. Challenge sites were evaluated 24, 48 and 72 h after challenge irradiation. The positive control substance (3, 3', 4', 5-tetrachlorosalicylanilide) tested in 10 animals provoked the adequate response. There was no indication of a sensitizing or photosensitizing potential observed in guinea pigs (Ciba Specilaity Chemical Corp., 2001). 

The test is adequate for assessment as it fulfils the requirements of the OECD draft guideline on the acute dermal photo-irradiation dose-response test of February 1995 and also the “Guidelines for Evaluation Photo-dermatitis” by the Cosmetic, Toiletry and Fragrance Association (CTFA) of 1991. In addition, the test was performed under GLP.


Short description of key information:
In the key study, only 5% of the test animals gave positive results after challenge exposures with the test article. Based on the evaluation criteria of Annex I of Regulation No. 1272/2008, the evaluation "sensitizing" is warranted when at least 30 % of the test animals exhibit positive skin reactions in an adjuvant test. Therefore, in line with the above argument, the test article is considered not sensitizing after skin contact.

Contact sensitization was absent in human volunteers whose skin were challenged with the test substance after a 3 week induction period. 0 out of 59 volunteers had positive responses.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No local reactions were observed in a guinea pig maximization test (OECD 406) after an intradermal induction with 5 %. Therefore, the substance does not require classification as a skin sensitizer under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.