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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from December 16, 1997 to January 23, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Valid results of the OECD 406 study were already available prior to development of the LLNA test protocol. Therefore, for animal welfare reasons an additional LLNA study is not performed.

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N,N',N'-tetrakis(2-hydroxypropyl)adipamide
EC Number:
260-982-3
EC Name:
N,N,N',N'-tetrakis(2-hydroxypropyl)adipamide
Cas Number:
57843-53-5
Molecular formula:
C18H36N2O6
IUPAC Name:
N,N,N',N'-tetrakis(2-hydroxypropyl)hexanediamide
Test material form:
other: solid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Himalayan
Sex:
female
Details on test animals and environmental conditions:
Conditions: Standard Laboratory Conditions Air-conditioned with 10 - 15 air changes per hour and continuously monitored environment with a target range for room temperature of 22 ±3 °C and for relative humidity between 40 - 70% (values above 70% during cIeaning process possible). The animals were provided with a 12-hour light, 12-hour dark cycIe. Music was played during the light period.
Accommodation: Individually in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz).
Diet: Pelleted standard Nafag Ecosan 845 25W4, batch nos. 102/97 and 112/97 guinea pig breeding / maintenance diet ("Nafag",Nähr- und Futtermittel AG, CH-9202 Gossau), ad libitum. Results of analyses for contaminants are archived at RCC.
Water: Community tap water from Füllinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (approx. 1 g/l) via the drinking water was provided. Results of bacteriological, chemical and contaminant analyses are archived at RCC.
Test system: Ibm: GOHI; SPF-quality guinea pigs (synonym: Himalayan spotted)
Rationale: Recognized by the international guidelines as a recommended test system (e.g. OECD, EEC).
Source: BRL, Biological Research Laboratories Ltd., WoIferstrasse 4, CH4414 Füllinsdorf / Switzerland
Number of animals for main study / pre-test: 15 females / 3 females, nulliparous and non-pregnant
Age at delivery: 4 - 6 weeks
Age at pre-test/beginning of acclimatization period: 4 - 6 weeks
Body weight at pre-test start: Pre-test groups: 333 - 374 g
Body weight at beginning of acclimatization period: Control and test group 313 – 406 g
Identification: By unique cage number and corresponding ear tags.
Randomization:Randomly selected at time of delivery.
Acclimatization: One week for the control and test group under test conditions after health examination. No acclimatization for the animals of the pre-test. Only animals without any visible signs of iIIness were used.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Remarks:
bi-distilled water
Concentration / amount:
induction:
intradermal application: 5% dilution of the test in bi-distilled water
epidermal application: 75% dilution of the test in bi-distilled water
challenge:
epidermal application: 25% dillution of the test in bi-distilled water
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Remarks:
bi-distilled water
Concentration / amount:
induction:
intradermal application: 5% dilution of the test in bi-distilled water
epidermal application: 75% dilution of the test in bi-distilled water
challenge:
epidermal application: 25% dillution of the test in bi-distilled water
No. of animals per dose:
The animals were distributed as follows:
NUMBER OF ANIMALS ANIMAL NUMBERS PER GROUP PERGROUP
1 Control Group 5 980 - 984
2 TestGroup 10 985 - 994
3 Intradermal Pre-test 1 995
4 Epidermal Pre-test 2 996 - 997
Details on study design:
The test article and vehicle were placed into a glass beaker on a tared Mettler PM 460 balance and a weight by weight dilution was prepared. Homogeneity of the test article in a suitable vehicle was maintained during treatment using a magnetic stirrer. The preparations were made immediately prior to each dosing.
Erythema and oedema were assessed using the following numerical grading system according to Draize (Draize J.H., Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics, Association of Food and Drug Officials of the United States, Austin, Texas, 1959).
Pre-test I performed during the acclimatization period:
INTRADERMAL INJECTIONS: Four intradermal injections (0.1 ml/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of one guinea pig. One week later intradermal injections (0.1 ml/site) were made into the clipped flank of the same guinea pig at concentrations of 5, 3 and 1% of the test article in bi-distilled water.
The resulting dermal reactions were assessed 24 hours later. For intradermal induction application in the main study a 5% test article concentration was selected.
Epidermal Applications: Four intradermal injections (0.1 ml/site) of a 1: 1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. One week later both flanks of each of the guinea pigs were clipped and shaved just prior to the application. Thereafter, 4 patches of filter paper (2 x 2 cm) were saturated with the test article at A = 75% (this concentration was found to be the most qualified to assure an optimum technical application procedure), B = 50%, C = 25% and D = 15% in bi-distilled water and applied to the clipped and shaved flanks. The volume of test article applied was approximately 0.2 g for the concentration of 75% and 0.2 ml for the remaining concentrations. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. This procedure ensured the intensive contact of the test article. The dressings were removed after an exposure period of 24 hours.
MAIN STUDY
INDUCTION
Intradermal injections / performed on test day 1: An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 ml/site) were made at the border of a 4 x 6 cm area in the clipped region.
Epidermal applications / performed on test day 8: On test day 8, a 2 x 4 cm patch of filter paper was saturated with the test article (75% in bi-distilled water) and placed between the injection sites of the test animals. The volume of test article applied was approximately 0.3 g. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The dressings were left in place for 48 hours. The epidermal application procedure described ensured intensive contact of the test article. The guinea pigs of the control group were treated as described above with bi-distilled water only. The volume applied was approximately 0.3 ml. Reaction sites were assessed for erythema and oedema 24 and 48 hours after removal of the dressing, using the numerical grading system according to Draize.
Challenge / performed on test day 22: The test and control guinea pigs were challenged two weeks after the epidermal induction application. The test and control guinea pigs were treated in the same way. Hair was clipped and shaved from a 5 x 5 cm area on the left and right flank of each guinea pig just prior to the application. Two patches (2 x 2 cm) of filter paper were saturated with the highest non-irritating concentration of 25% (left flank) and the vehicle only (bi-distilled water applied to the right flank) using the same method as for the epidermal application. The volume of test article applied was approximately 0.2 mL The dressings were left in place for 24 hours.
Approximately 21 hours after removal of the dressing the test sites treated with the test article were depilated as described in the epidermal pre-test.
Approximately 24 and 48 hours after the removal of the dressing the application sites were assessed for erythema and oedema using the numerical scoring system according to Draize.

Challenge controls:
no data
Positive control substance(s):
yes
Remarks:
2-MERCAPTOBENZOTHIAZOLE

Results and discussion

Positive control results:
The intradermal induction of sensitization was carried out with a 5% dilution of the test article in mineral oil and in an emulsion with Freund's Complete Adjuvant (FCA) / physiological saline. The epicutaneous induction of sensitization was conducted under occlusion with the test article at 50% in mineral oil. Two weeks after the epicutaneous induction application the challenge was completed by epicutaneous application of the test article at 1% in mineral oil under occlusive dressing. The animals of the control group were induced with mineral oil and FCA/physiological saline and challenged similarly to those of the test group. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 hours after removal of the dressing.
In this study 50% of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test article concentration of 1% in mineral oil. No skin reactions were observed in the control group. A response of at least 30% positive animals is considered positive "R43": may cause sensitization by skin contact according to the "Commission Directive 96/54/EEC, July 30, 1996 adapting to technical progress for the 22nd time Council Directive 67/548/EEC on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances. Therefore, the test article 2-MERCAPTOBENZOTHIAZOLE applied at a concentration of 1% in mineral oil is considered to be a sensitizer when used under the described test conditions.
According to the rating of allergenicity by Magnusson and Kligman 2-MERCAPTO-BENZOTHIAZOLE is considered to be a moderate sensitizer.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
5% dilution in mineral oil and in FCA/physiological saline
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
No deaths and no toxic symptoms
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
5% dilution in mineral oil and in FCA/physiological saline
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
No deaths and no toxic symptoms
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
test article
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no adverse effect
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: test article. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no adverse effect.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
bi-distilled water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no adverse effect
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no adverse effect.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
test article
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no adverse effect
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: test article. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no adverse effect.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no adverse effect
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no adverse effect.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
bi-distilled water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no adverse effect
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no adverse effect.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
test article
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no adverse effect
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: test article. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no adverse effect.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
bi-distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no adverse effect
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: bi-distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no adverse effect.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
test article
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no adverse effect
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: test article. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no adverse effect.

Any other information on results incl. tables

The expected local symptoms were observed in the animals of the control and test group after the intradermal injections.


CONTROL GROUP: No erythematous or oedematous reaction was observed in the animals treated with bi-distilled water only.


TEST GROUP: No erythematous or oedematous reaction was observed in the animals treated with the test article at 75% in bi-distilled water.


SKIN EFFECTS AFTER THE CHALLENGE - PERFORMED ON TEST DAY22


CONTROL AND TEST GROUP: No positive reactions were observed in the animals either when treated with bi-distilled water only or when treated with the test article at 25% in bi-distilled water.


As there were no deaths during the course of the treatment period no necropsies were performed.


No symptoms of systemic toxicity were observed in the animals.


A loss of body weight was observed in two control animals and one test animal during the acclimatization phase. One animal of the epidermal pre-test lost weight during its treatment phase.


The body weight of the other animals was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
In this study none of the animals of the test group were observed with positive skin reactions after treatment with a non-irritant test article concentration of 25% in bi-distilled water. No skin reactions were observed in the control group. Therefore, the test article applied at a concentration of 25% in bi-distilled water is considered not to be a skin sensitizer when used under the described test conditions.
Executive summary:

The intradermal induction of sensitisation was performed with a 5% dilution of the test article in bi-distilled water and in an emulsion with Freund's Complete Adjuvant (FCA) / physiological saline. The epidermal induction of sensitisation was conducted under occlusion with the test article at 75% in bi-distilled water. Two weeks after the epidermal induction application the challenge was completed by epidermal application of the test article at 25% in bi-distilled water under occlusive dressing. The animals of the control group were induced with bi-distilled water and FCA/physiological saline and challenged similarly to those of the test group. Cutaneous reactions, i.e. erythema and eschar, as well as oedema formation were evaluated at 24 and 48 hours after removal of the dressing.


No toxic symptoms were evident in the guinea pigs of the control or test group. No deaths occurred.


Therefore, it is concluded that test substance is not considered to be a sensitiser to skin of guinea pigs.