Registration Dossier

Administrative data

Description of key information

The substance is included in the category of Stilbene Fluorescent Whitening Agents (SFWA) comprising of 14 member substances in total. The category of Stilbene Fluorescent Whitening Agents is defined as a structurally related group of substances that are derivatives of 4,4’-bis(1,3,5-triazinyl-2-yl)amino)stilbene-2,2’-disulphonic acid, each with one aniline and one alkyl derivative amino moiety at the triazine ring. The aniline moiety can also be mono- or di-sulfonic aniline. With the exception of 3a-A (free acid) and OB 3a-A (NaK) all substances are sodium salts. The molecular structures of 3a-A (free acid), OB 3a-A (NaK) and OB 3a-A (Na) are the same except that the first one is the free sulfonic acid and the second one the potassium/sodium salt. The chemical structures are highly symmetric with an extended chromophor system. Substituents have little influence on the chromophor system; they affect the application properties and substantivity. To be included in the defined category of SFWA a substance must have the following characteristics:


 •           All substances are derivatives of 4,4’-bis(1,3,5-triazinyl-2-yl)amino)stilbene-2,2’-disulphonic acid, each with one aniline derivative (R1) and one alkyl derivative amino moiety at the triazine ring (R2).


•            The substances have all negative calculated log Pow (< -1), due to the fact that except from the acid form, they are all salts.


•            Molecular weights range from ca. 872 to ca. 1369 g/mol.


•            All category members are very soluble, with an individual solubility ranging from ca. 1 to >200 g/l. Water solubility tends to increase with the degree of sulfonation. They are all very stable and do not hydrolyze.


•            As a result of the stilbene portion of the molecule, common to all category members, these fluorescent whitening agents have an UV absorption maximum between 340 to 360 nm in water, which makes them subject to direct photo-degradation in the hydrosphere.


The structural differences between the substances are determined by the presence of two moieties (R1 and R2) bound to both triazine rings. Each substance is characterized by a combination of two of these moieties to each triazine ring and by the cations of the sulfonic acid group present, which in almost all cases is Na+, in one case it is Na+ and K+; instead of these cations a proton (H+) is present for one substance. R1 is bound to the 3 position of the triazine ring and is described as an aniline function, which may have one or two sulfonate functions.


Based on the variability related to R1 or R2 two different sub-categorizations can be considered - Sub-categorization based on R1 or Grouping based on R2. R2 can be constituted by the following:


•            morpholino


•            methyl (2-hydroxyethyl)amino,


•            2-hydroxyethylamino,


•            bis(2-hydroxyethyl)amino,


•            diethylamino


•            (2-carbamoylethyl)(2-hydroxyethyl)amino,


•            bis(2-hydroxypropyl)amino,


For read-across both of these structural differences in moiety R1 and R2 are taking into account. Therefore read-across to OB 2 -A having an identical structure in moiety R2 and to OB 3a-DSA having an identical structure in R1 is applied. Skin sensitization is a systemic endpoint; therefore skin absorption is required to express the effect. Dermal absorption is estimated to be negligible for all members of the category based on physicochemical properties (see Table 19 of the category justification) and also measured for OB 2 -A and OB 3b-A. No dermal metabolism is indicated with the OECD QSAR Toolbox. The available data on the substances in the category show that they are all negative regarding skin sensitization. Therefore, based on structural similarities, lack of specific alerts, and predicted specific endpoint results, a read across approach for the substances that have not been tested is justified, hence as all results were consistently negative there is no evidence for skin sensitization properties of any category member.


 


Justification for read across:


1)     The substance is part of the Group 2, in which the members share the common organic functional group morpholino derivative, with different sulfonation degrees: bisulfonated (OB 2 -A) tetrasulfonated (OB 2 -MSA) and hexasulfonated (OB 2 -DSA).


2)     The studies performed on OB 1 -DSA, harvesting in organic functional group diethylamino derivative instead of the morpholino derivative but sharing the same residue in R1, are also considered as supporting reference. Both substances (OB 1 -DSA and OB 2 -DSA) are hexasulfonated sodium salts, very similar according to Tanimoto rules.


3)     Within the whole category no substance showed concern about skin sensitization potential.


4)     OECD QSAR Toolbox previsions suggest no skin sensitization potential for the substance, based on no alert found for protein binding by OASIS v. 1.2, protein binding by OECD, protein binding potency, and protein binding alerts for skin sensitization.


5)     Skin sensitization is a systemic effect and dermal adsorption needs to provide the effect. Very low dermal absorption based on predicted values calculated with DERMWIN and confirmed by measured dermal absorption on a similar substances of the category (OB 3b-A).


6)     No dermal metabolism has been proposed by the dermal metabolism simulator in the OECD QSAR Toolbox.


 


In summary, OB 2 -DSA is expected to have no skin sensitization potential which is in line with results obtained in various in vivo tests among the category members. Therefore, also with respect to animal health and welfare, read-across to OB 2 -A and OB 2 -MSA is applied and no further testing in vivo is considered.

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
Justification for type of information:
Read-across to the target chemial is claimed based on the category of stilbene optical brighteners as documented within the category justification report attached to Chapter 13 of this dossier.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Remarks:
RCC AG, Itingen, Switzerland
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Data from a reliable in vivo test conducted before the enforcement of Commission Regulation (EU) 640/2012 of 06 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) are available.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf / Switzerland
- Age at study initiation: males: 7 weeks, females: 8 weeks
- Weight at study initiation: males: 336 - 461 g, females: 332 - 428 g
- Housing: single
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: one week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route:
intradermal and epicutaneous
Vehicle:
other: distilled water and petrolatum oil
Concentration / amount:
intradermal induction: 5 %
cutaneaous induction: 25 %
challenge: 10 %
Route:
epicutaneous, occlusive
Vehicle:
other: distilled water and petrolatum oil
Concentration / amount:
intradermal induction: 5 %
cutaneaous induction: 25 %
challenge: 10 %
No. of animals per dose:
control group: 5 male and 5 female
test group: 10 male and 10 female
Details on study design:
RANGE FINDING TESTS: The objective of this investigation was to identify irritant test article concentrations suitable for the induction phase of the main study. In addition, a suitable non-irritant concentration of the test article, by the topical route of administration, was identified for the challenge application. The procedure applied for these investigations was as follows:
Intradermal injections: Intradermal injections (0.1 ml/site) were made into the clipped flank of two guinea-pigs at concentrations of 1, 3 and 5 % of the test article in distilled water. The resulting dermal reactions were assessed 24 hours later.
Epidermal applications: Patches of filter paper ( 2 x 2 cm) were saturated with concentrations of 3, 5, 10 and 25 % of the test article in petrolatum oil and applied to the clipped and shaved flanks of each of four guinea-pigs. The patches were covered by a strip of aluminum foil and firmly secured by elastic plaster wound round 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 and the reaction sites were assessed for erythema and edema on a numerical basis according to the scale described above. Further examination of the sites were performed 24 and 48 hours after removal of the dressings.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (one intradermal, one epidermal)
- Exposure period: 1 week after intradermal, 48 hours epidermal application
- Test groups: 5 % intradermal application, 25 % epidermal application
- Control group: intradermal with distilled water, epdermal with petrolatum oil
- Site: scapular region


B. CHALLENGE EXPOSURE
- No. of exposures: 1 epidermal application
- Day(s) of challenge: two weeks after epidermal induction
- Exposure period: 24 hours
- Test groups: 10 % test article in petrolatum oil, and vehicle alone
- Control group: 10 % test article in petrolatum oil, and vehicle alone
- Site: right and left flank
- Concentrations: 10 %
- Evaluation (hr after challenge): 24 and 48 hours after challenge
Positive control substance(s):
yes
Remarks:
1-chloro-2,4-dinitro-benzol (DNCB)
Positive control results:
For the induction period a 0.5 % dilution of DNCB in ethanol, and for the challenge procedure a 0.3 % dilution of DNCB was used.
Positive erythema reaction after first challenge procedure after 24 hours:
postive: 6 animals; negative: 3 animals
67 % with positive reaction
According to the results observed it is considered that DNCB possess a strong skin sensitizing (contact allergenic) potential in the guinea pig strain used (Dunkin-Hartley albino guinea pigs; DUHA KFM. Kleintierfarm Madoerin AG, CH 4414 Fuellinsdorf / Switzerland).
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10%
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. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10%
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. Dose level: 10 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10%
No. with + reactions:
1
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: 10 %. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10%
No. with + reactions:
1
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: 10 %. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.3%
No. with + reactions:
6
Total no. in group:
9
Remarks on result:
positive indication of skin sensitisation
Conclusions:
According to the results described above a weak allergenic potency of the test article was observed in this test when followed the rating of allergenicity described by Magnusson B. and Kligman A.M. (1969). According to EEC (European Economic Community) classification criteria described in guidelines 83/467, September 16, 1983 however, this test article is not a sensitizer.
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
Justification for type of information:
Read-across to the target chemial is claimed based on the category of stilbene optical brighteners as documented within the Category justification report attached to Chapter 13 of this dossier.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Remarks:
RCC, Research & Consulting Company AG
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Data from a reliable in vivo test conducted before the enforcement of Commission Regulation (EU) 640/2012 of 06 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) are available.
Species:
guinea pig
Strain:
other: Ibm:GOHI (SPF)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd. Wolferstrasse 4, CH-4414 Fullinsdorf
- Age at study initiation: 8 weeks
- Weight at study initiation: 396 - 432 g
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding
- Diet: Pelleted standard Kliba 342, Batch 61/90 guinea pig breeding/ maintenance diet (Klingentalmuhle AG, CH-4303 Kaiseraugst), ad libitum.
- Water: Community tap water from Fullinsdorf, ad libitum. Once weekly additional supply of ascorbic acid (1g/l) via the drinking water.
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature: 22±3 °C
- Humidity (%): 40-70 %
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route:
intradermal and epicutaneous
Vehicle:
other: intradermal: physiological saline, epicutaneous: 25 % in vaselinum album
Concentration / amount:
1st: Induction 1 % intracutaneous,
2nd: Induction 25 % occlusive epicutaneous,
3rd: Challenge 25 % occlusive epicutaneous.
Route:
epicutaneous, semiocclusive
Vehicle:
other: intradermal: physiological saline, epicutaneous: 25 % in vaselinum album
Concentration / amount:
1st: Induction 1 % intracutaneous,
2nd: Induction 25 % occlusive epicutaneous,
3rd: Challenge 25 % occlusive epicutaneous.
No. of animals per dose:
control group: 10
test group: 20
pretest: 6
Details on study design:
RANGE FINDING TESTS:
Intradermal injections (0.1 ml/site) were made into the clipped flank of 2 guinea pigs at 1, 3, and 5%. The resulting dermal reactions were scored 24 hours later. Patches of filter paper (2 x 2 cm) were covered with a thin layer of test material in vaselinum album at 5, 10, 15 and 25 % and applied to the clipped and shaved flanks of each of 4 guinea pigs. The patches were covered with a strip of aluminum foil and firmly secured by elastic plaster wrapped around the trunk and covered with impervious adhesive tape. The dressings were removed after 24 hours and the reactions were scored immediately and 24 and 48 hours later.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal: 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. Test animals were injected with Freund's complete adjuvant:physiological saline (50:50), 1 % test material (in physiological saline) or test material diluted to 1 % by emulsion in a 50:50 mixture of Freund's complete adjuvant and physiological saline at the 3 sites. Control animals were injected with Freund's complete adjuvant:physiological saline (50:50), physiological saline, or a 50:50 mixture of Freund's complete adjuvant and physiological saline.

Epidermal: On day 7 of the test (approximately 24 hours prior to epidermal application), the scapular area (approximately 6 x 8 cm) was clipped, shaved free of hair and pretreated with 10 % sodium-lauryl-sulfate (SLS) in petrolatum oil, because none of the concentrations given previously in the pretest (up to 25 %) caused irritation. The SLS was massaged into the skin with a glass rod without bandaging. The treatment provoked a mild inflammatory reaction. A day later, a 2 x 4 cm patch of filter paper was covered with a thin layer of the selected test material concentration (25 % in vaselinum album) and placed over the injection sites of the test animals. The patch was covered by aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animals and secured with impervious adhesive tape. The dressings were left in place for approximately 48 hours. The control group was treated similarly with the omission of the test material. Reaction sites were assessed for erythema and edema immediately, and 24 and 48 hours after removal of the dressing.


B. CHALLENGE EXPOSURE
Challenge: Test and control animals were challenged 2 weeks after the epidermal induction and application. Hair was clipped from a 5 x 5 cm area on the left and right flank of each animal. Two patches (2 x 2 cm) of filter paper were covered with a thin layer of a non-irritant concentration of test material (25 % in vaselinum albumin) and with vaselinum album only, applied to the left and right flank using the same method as for the epidermal application. The dressings were removed approximately 18 hours later. The sites were assessed for erythema and edema immediately, and 24 and 48 hours after removal of the dressings. Control animals were treated similarly, omitting the test material. All animals were euthanized at the end of the test with an i.p. injection of pentobarbital (>800 mg/kg).
Positive control substance(s):
yes
Remarks:
potassium dichromate
Positive control results:
For the induction period a 10 % dilution of potassium dichromate in physiological saline and for the challenge procedure a 2.5 % dilution of potassium dichromate in physiological saline was used. According to the results observed (8/10 positive) it is considered that the known allergen potassium dichromate possess a strong skin sensitizing potential in the guinea pig strain used.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. Hours after challenge: 24.0. Group: negative control. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. Hours after challenge: 48.0. Group: negative control. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
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.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25%
No. with + reactions:
0
Total no. in group:
20
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.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
2.5%
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. Hours after challenge: 24.0. Group: positive control. Dose level: 2.5 %. No with. + reactions: 8.0. Total no. in groups: 10.0.

No positive reactions were evident after the first challenge application, neither when treated with vaselinum album only nor when treated with a 25 % test article dilution.

Intradermal injection: During the pretest, all scores for erythema and edema were 2 (well defined) at all concentrations (with the exception one animal treated with 1 % that had a score of 1 [barely perceptible] for erythema). According to the findings observed, the concentration selected for the main study was 1 %. Epidermal application: All concentrations tested produced erythema scores of 1 (barely perceptible) immediately after exposure, with the exception that 5 and 10 % produced scores of 0 in one animal at this time. All other values were 0. Based on the findings, 25 % was selected for induction and challenge.

 

Main study:

Induction: All ten control animals had erythema scores of 1 (barely perceptible) immediately after removal of the bandage. One had an edema score of 1 immediately after removal of the bandage. This same animal had an erythema score of 1, 24 hours after removal. All other control scores were 0. Nine out of twenty test animals had erythema scores of 1 (barely perceptible) immediately after removal of the bandage. One test animal had an erythema score of 2 at this time. Seven out of 20 test animals had edema scores of 1 immediately after removal of the bandage. Six and two test animals had erythema scores of 1, 24 and 48 hours after bandage removal (respectively). One animal had an edema score of 1 at 24 and 48 hours.

 

Challenge: The material was not sensitizing. All controls challenged with the vaselinum album had erythema scores of 1 immediately after bandage removal. All other control scores were 0. Nine out of 10 controls challenged with test material (25%) in vaselinum album were 1 immediately after bandage removal. All other scores were 0. Nineteen out of 20 animals induced with test material and challenged with vaselinum album had scores of 1 immediately after bandage removal. All other scores in this group were 0. All 20 animals induced and challenged with test material had scores of 1 immediately after bandage removal. All other scores in this group were 0.

 

Other: None of the animals died. No clinical signs were observed. Body weight gain of animals was not affected by treatment.

 

Interpretation of results:
other: not sensitising
Conclusions:
The substance is not sensitizing to skin.
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:
other: Non standard test with adequate description.
Justification for type of information:
Read-across to the target chemial is claimed based on the category of stilbene optical brighteners as documented within the category justification report attached to Chapter 13 of this dossier.
Principles of method if other than guideline:
Maurer optimisation test
GLP compliance:
no
Type of study:
Maurer optimisation test
Justification for non-LLNA method:
Data from a reliable in vivo test conducted before the enforcement of Commission Regulation (EU) 640/2012 of 06 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) are available.
Species:
guinea pig
Strain:
Pirbright-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: own breed
- Weight at study initiation: 350 to 400 g
- Housing: individually in Macrolon cages, type 3
- Diet: standard guinea pig pellets - NAFAG No. 830, Gossau SG - and fresh carrots, ad libitum
- Water: water ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±1 °C
- Humidity (%): 55±5 %
- Photoperiod (hrs dark / hrs light): 10/14


Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
1st: Induction 0.1 ml of 0.1 % dilution intracutaneous,
1st: Challenge 0.1 ml of 0.1 % dilution intracutaneous,
2nd: Challenge non-irritant concentration (not further specified) occlusive epicutaneous
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
1st: Induction 0.1 ml of 0.1 % dilution intracutaneous,
1st: Challenge 0.1 ml of 0.1 % dilution intracutaneous,
2nd: Challenge non-irritant concentration (not further specified) occlusive epicutaneous
No. of animals per dose:
20
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0.1 %
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.1 %. No with. + reactions: 0.0. Total no. in groups: 20.0.

No difference between the test and the control group was found after epidermal challenge.

Interpretation of results:
other: not sensitising
Conclusions:
The substance is not sensitizing to skin.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 22 to May 14, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Read-across to the target chemial is claimed based on the category of stilbene optical brighteners as documented within the category justification report attached to Chapter 13 of this dossier.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands.
- Age at study initiation: 8 - 12 weeks (beginning of treatment).
- Housing: single
- Cage Type: Makrolon Type I, with wire mesh top.
- Bedding: granulated soft wood bedding.
- Diet: pelleted standard diet, ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: at least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: relative humidity 30-70 %
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.
Vehicle:
dimethylformamide
Remarks:
purity: 99 %
Concentration:
0 (vehicle group), 5, 10, 25 %
No. of animals per dose:
4 animals per dose group.
Details on study design:
RANGE FINDING TESTS
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 25 % suspension in dimethylformamide. Warming and sonicating could not achieve a higher concentration. With other vehicles used, e.g actone:olive oil (4+1), methyl ethyl ketone, DMSO, ethanol:deionised water (7+3) or propylene glycol, higher concentrations could also not be achieved. Upon sponsor's consent dimethylformamide was used as a vehicle.

To determine the highest non-irritant test concentration, a pre-test was performed in two animals. Two mice were treated with concentrations of 2.5, 5, 10, and 25 % on one ear each on three consecutive days. Clinical signs were recorded 24 ± 4 hours after each application. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity. The test item in the main study was assayed at 5, 10, and 25 %. The top dose is the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY
TOPICAL APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5, 10, and 25 % (w/v) in dimethylformamide. The application volume, 25 µl, was spread over the entire dorsal surface (0 ~ 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION of ³H-Methyl Thymidine
Five days after the first topical application, all mice were administered with 250 µl of 78.9 µCi/ml ³HTdR (corresponds to 19.7 µCi ³HTdR per mouse) by intravenous injection via a tail vein.

DETERMINATION of INCORPORATED ³HTdR
Approximately five hours after treatment with ³HTdR all mice were euthanised by intraperitoneal injection of Pentobarbital-Natrium (Release®, WOT, 0-30827 Garbsen). The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group).
Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 1 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.
The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to plastic scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid (Perkin Elmer (LAS) GmbH, 0-63110 Rodgau) and thoroughly mixed.
The level of ³HTdR incorporation was then measured on a β-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, 0-63110 Rodgau). Similarly, background ³HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute (OPM).

OBSERVATIONS
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: once daily (week day) from experimental start to necropsy.
Body weights: prior to the first application and prior to treatment with ³HTdR.
Clinical signs (local / systemic): once daily (week day). Especially the treatment sites were observed carefully.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
EC3 = 15.7 % (w/v)
Parameter:
SI
Value:
ca. 0.64
Test group / Remarks:
4 animals - Dose 5%
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM measured Background I: 29 Background II: 30 Control: 5370 Group 2: 3446 Group 3: 6695 Group 4: 4612
Parameter:
SI
Value:
ca. 1.25
Test group / Remarks:
4 animals - Dose 10 %
Parameter:
SI
Value:
ca. 0.86
Test group / Remarks:
4 animals - Dose 25 %

Vehicle: dimethylformamide








































































Test item conc. % (w/v)GroupMeasurement
DPM
CalculationResult
DPM-BG*number of lymph nodesDPM per lymph nodes**S.I.
-BG I29----
-BG II30----
-1537053418667.6 
52344634178427.100.64
103669566668833.201.25
254461245838572.800.86

BG = Background (1 ml 5 % trichloroacetic acid) in duplicate


1 = Control Group


2-4 = Test Group


S.I. = Stimulation Index


* = The mean value was taken from the figures BG I and BG "


** = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.


The EC3 value could not be calculated, since all S.I.'s are below 3.


Other results


Viability/Mortality: no deaths occurred during the study period.


Clinical Signs: the animals did not show any clinical signs during the course of the study and no cases of mortality were observed.


Body Weights: the body weight of the animals, recorded prior to the first application and prior to treatment with ³HTdR, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:
other: not sensitising
Conclusions:
The test item test item was found to be not a skin sensitizer under the described conditions.
Executive summary:

Method

In the study the test item suspended in dimethylformamide was assessed for its possible contact allergenic potential, according to the OECD Guideline 429 Skin Sensitisation: Local Lymph Node Assay (adopted 24 April 2002). For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25 %.

Results

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 0.64, 1.25, and 0.86 were determined with the test item at concentrations of 5, 10, and 25 % in dimethylformamide, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

Conclusion

The test item test item was found to be not a skin sensitizer under the described conditions.

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)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance was considered to be a non-sensitiser, hence, does not warrant classification as per the CLP (Regulation EC No. 1272/2008) criteria.