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Diss Factsheets

Administrative data

Description of key information

 OECD 429, the substance is not skin sensitising.


 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose for cross-reference:
data waiving: supporting information
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
in vivo
Type of information:
other: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Source study has reliability 1. Details on the read across are attached in section 13.
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Directive 92/69 EEC B6
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Guinea pig maximisation test was available
Species:
guinea pig
Strain:
Himalayan
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wölferstrasse 4, 4414 Füllinsdorf/Switzerland
- Age at study initiation: 5-8 weeks
- Weight at study initiation: control and test group: 341-489 g. Pretest 317-435 g
- Housing: individually
- Diet: pelleted standard Kliba 342, Batch no 68/95 guinea pig breeding/maintenance diet, ad libitum
- Water: community tap water from Füllinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1 g/l) via the drinking water.
- Acclimation period: one week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pretest. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 21-23 °C
- Humidity: 48-60 %
- Air changes: 10-15 per hr
- Photoperiod: 12 hour light, 12 hour dark cyle

Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
Test:
FCA : phys. saline 1:1
5 % bi-distilled water
5 % FCA : phys. saline 1:1
Control:
FCA : phys.saline 1:1
bi-distilled water
FCA : phys. sal. 1:1/bi-distilled water
Day(s)/duration:
day 1
Route:
epicutaneous, semiocclusive
Vehicle:
physiological saline
Concentration / amount:
- 25 % bi-distilled water
Day(s)/duration:
day 8
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
physiological saline
Concentration / amount:
15 % bi-distilled water
Day(s)/duration:
day 22
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Control group: 10 males
Test group: 20 males
Intradermal pretest: 2 males
Epidermal pretest: 4 males
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: three pairs of intradermal injections
- Exposure period: 7 days
- Test groups:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
2) test article, diluted to 5 % with bi-distilled water
3) test article diluted to 5 % by emulsion in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

- Control group:
1) 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
2) bi-distilled water
3) 1:1 (w/w) mixture of bi-distilled water in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline.

- Site: dorsal skin from the scapular region (approximaterly 6 × 8 cm) was clipped free of hair.
- Frequency of applications: 1 week
- Duration: 48 h


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 22
- Exposure period: 24 - 48 h
- Site: hair was clipped and shaved from a 5 × 5 cm area on the left and right flank of each guinea-pig just prior to the application.
- Concentrations: 2 patches ( 2 × 2 cm) of filter paper were saturated with the highest non-irritating concentration of 15 % (left flank) and the vehicle only (bi-distilled water, applied to the right flank) using the same method as for the epidermal application.
- Duration: 24 h
- Evaluation: 24 and 48 h after removal of the dressing
Positive control substance(s):
yes
Remarks:
4-aminobenzoic acid ethyl ester and 2- mercaptobenyothiazol
Positive control results:
For validation of sensitivity, a known sensitizer (2-mercaptobenzothiazol) was selected as a positive control.
In this study, 95 % and 90 % of animals of test group were observed with positive skin reactions at the 24- and 48-hour reading respectively after treatment with a non-irritant test substance concentration of 5 % in peanut oil. No skin reactions were observed in the control group. The results obtained with test article at 10 % in peanut oil were not taken into consideration since both control group were observed with erythematous reactions.
A known mild sensitizer was also selected as a positive control (4-aminobenzoic acid ethyl ester). In this study 30 % and 35 % of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test substance concentration of 30 % in mineral oil. No skin reactions were observed in the control group.
The response of at least 30 % positive animals is considered positive.
Test article 2-mercaptobenzothiazol at concentration of 5 % in peanut oil is considered an extreme sensitiser.
Test article 4-aminobenzoic acid ethyl ester at concentration of 30 % in mineral oil is considered a moderate sensitiser.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
15 % in bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
15 % in bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle, bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
15 % in bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
erythem and edema
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
15 % in bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
erythema and edema
Remarks on result:
no indication of skin sensitisation

After epidermal induction, due to orange-red discoloration produced by test substance, a possible erythema reaction could not be determined.

Viability / mortality / macroscopic findings: as there were no deaths during the treatment period, no necropsies were performed.

Clical signs, systemic: no symptoms of systemic toxicity were observed in the animals.

Body weight: 2 out of 10 animals of the control group (nos. 325, 330) and 1 out of 20 animals of the test group (no. 346) incidentally lost weight during the acclimatisation period. No weight loss was observed during treatment period.

Interpretation of results:
other: not classified, according to the CLP Regulation (EC 1272/2008)
Conclusions:
The substance was tested according to the guidelines OECD 429
On the base of results, the substance was considered as not skin sensitising.
Executive summary:

Method

A guinea pig maximisation test was carried out to assess the allergenic potential of the substance. Ten males were used as control group and 20 males were used as test group.

Before the main test, a pretest was performed in order to identify a maximally tolerated concentration of test article suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of test article, by the topical route of administration, was identified for the challenge application.

The pretest was performed by intradermal (0.1 ml/site, at concentrations of 1, 3 and 5 % of test article in bi-distilled water) and epidermal route (5, 10, 15 and 25 % of test material in bi-distilled water).

Based on pretest findings:

- application a 5 % test substance dilution in bi-distilled water was selected for intradermal induction

- 25 % test substance in bi-distilled water was selected for the induction application and 15 % test substance in bi-distilled water was selected for the challenge application.

Results

None of the animals of the test group showed positive skin reactions responding to a 5 % intradermal induction dose. Therefore, test substance was considered as not skin sensitising.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

No study on target substance was available, thus a read across approach was followed. In particular a guinea pig maximisation test carried out on Similar Substance 01 was used for the assessment. Details on the read across are attached in section 13.


 


In a study from 1995, no information on composition of test sample was available. However, test material was described as "purified", compared to a test sample described as "crude", with a content of 91 % of active substances. No sensitising responses were seen after 5 % intradermal induction.


In a study from 1993, the "crude" sample was tested and gave a positive response after 5 % intradermal induction. Based on the lower purity of the test sample compared to the sample used in the study from 1995, the sensitisation response was reasonably attributed to the impurities content.


 


The lack of sensitising potential was confirmed by a human repeated insult patch test from 1972. Test sample gave a negative result on sensitisation when tested at 4 % in 200 individuals covering a wide range of ages. 

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Category 1


Substances shall be classified as skin sensitizers in category 1 where data are not sufficient for sub-categorisation in accordance with the following criteria:


(a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons; or


(b) if there are positive results from an appropriate animal test.


As for guinea pig maximisation test, a response of at least 30 % of animals is considered positive.


 


Subcategorisation is done as follows in case of a guinea pig maximisation test:


Sub-category 1A


Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.


Specific criteria: ≥ 30 % responding at ≤ 0.1 % intradermal induction dose or ≥ 60 % responding at > 0.1 % to ≤ 1 % intradermal induction dose


 


Sub-category 1B


Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.


Specific criteria: ≥ 30 % to < 60 % responding at > 0.1 % to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % topical induction dose.


 


As no positive response was seen in test animals upon a 5 % intradermal induction dose, the substance is not classified within the CLP Regulation (EC 1272/2008).