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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 September to 10 November 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary guideline and GLP compliant study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This is the scientific justification for performing the OECD Test No. 406 for Skin Sensitisation, i.e. the Guinea Pig Maximization Test (GPMT), instead of1he required OECD Test No. 429, i.e. Skin Senitisation according to the Local Lymph Node Assay.

In recently published articles in peer reviewed journals. see reference list, it is clearly demonstrated that surfactants are more likely to give rise to false positives in the LLNA. Consequently, in the evaluation of such substances for sensitizing properties the LLNA test is not an appropriate assay and would not represent an optimum use of test animals. It is therefore recommended that the Guinea Pig Maximization Test (GPMT) is used instead. This is also supported by the TG OECD 406 ”In addition, test substance classes or substances containing functional groups shown to act as potential confounders (Basketter et al., 2009) may necessitate
the use of guinea pig tests".
References:
Kreiling. R .. Hollnagel, H..M ; Hareng. L.. Eigler D. .. Lee. M.S . Griem. P .. Dreesen B,
Klebcr. M ., Albrecht. A.. Garcia. C .• Wendel, A (2008) ,Comparison of the skin
sensitizing potential of unsaturated compounds and assessed by the murine
local lymph node assay (LLNA) and the guinca pig maximization test (GPMT).
Food Chem. Toxicol. 46. 1896 – 1904

D. Basketter. N. Ball. S. Cagen,. JC Carrillo. H. Certa. D. Eigler. H. Esch. C. Garcia, C. Graham. C.Haux. R.
Kreiling. A. Mehling. (2009) Application of a weight of evidence approach to assessing discordant sensitisation
datasets: Jmplications for REACH. Reg Tox Pharrn. 55:90-96.

C. Garcia. . N. Ball. S. Cagen,. JC Carrillo. H. Certa. D. Eigler. H. Esch C. Graham.. C.Haux, R. Kreiling. A.
Mehling. (2010) Comparative testing for the identification of skin sensitizing potentials of nonionic sugar lipid
surfactants. Reg Tox Pharrn 58: 301-307
Note: Ref list not complete due to lack of space.
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
The animals were male and nulliparous and non-pregnant female Hartley Crl: (HA) BR guinea pigs, Caesarian obtained, Barrier sustained - Virus Antibody Free (COBS - VAF). The guinea pigs were supplied by Charles River Laboratories, France, and acclimatised for at least 5 days. The guinea pigs were weighed and randomly allocated to groups the day before study initiation. On the first day of the study, the animals in the main test were 1-2 months old and had a mean body weight ± standard deviation of 366±17 g for the males and 321±16 g for the females. Individuals were identified by ear tattoo.
The animal rooom was maintained at a temperature of 22±2°C, relative humidity 30 to 70%, 12 hour light dark cycle with approximately 12 air changes per hour. During the acclimation period and throughout the study, the animals were housed individually in polycarbonate cages with stainless steel lid (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle. Each cage contained autoclaved sawdust (SICSA, France).
106 pelleted diet (SAFE, France) and filtered drinking water were provided ad libitum.
Route:
intradermal and epicutaneous
Vehicle:
other: The vehicle for intradermal injections was corn oil. For topical applications the vehicles were: induction - an 80/20 (w/w) mixture of ethanol and purified water; challenge - acetone.
Concentration / amount:
Preliminary test: induction intradermal injections of 25, 10, 5, 1 and 0.1 % (w/w); cutaneous induction concentrations of 100, 50, 25 and 10% (w/w); cutaneous challenge: 100, 50, 25, 10, 5 and 1% (w/w).
Main test: induction intradermal injections of 1% (w/w); cutaneous induction: 10% (w/w); cutaneous challenge 5 and 1% (w/w).
Route:
epicutaneous, occlusive
Vehicle:
other: The vehicle for intradermal injections was corn oil. For topical applications the vehicles were: induction - an 80/20 (w/w) mixture of ethanol and purified water; challenge - acetone.
Concentration / amount:
Preliminary test: induction intradermal injections of 25, 10, 5, 1 and 0.1 % (w/w); cutaneous induction concentrations of 100, 50, 25 and 10% (w/w); cutaneous challenge: 100, 50, 25, 10, 5 and 1% (w/w).
Main test: induction intradermal injections of 1% (w/w); cutaneous induction: 10% (w/w); cutaneous challenge 5 and 1% (w/w).
No. of animals per dose:
Preliminary test: 5 males and 5 females.
Main test: 10 males and 10 females in the test group; 5 males and 5 females in the control group.
Details on study design:
Preliminary test: A preliminary test was conducted in order to determine the concentrations to be tested in the main study.
By intradermal route (tested concentrations: 25%, 10%, 5%, 1% and 0.1% (w/w)): intradermal injections of the dosage form preparations (0.1 ml) were performed in the interscapular region, local reactions were evaluated approximately 24, 48 hours and 6 days after the injections.
By cutaneous route: under the conditions of the induction phase (tested concentrations: 100%, 50%, 25% and 10% (w/w)): a filter paper (approximately 8 cm2) was fully-loaded with a dosage form preparation and was then applied to the clipped area of the skin. The filter paper was held in place by means of an occlusive dressing for 48 hours, cutaneous reactions were evaluated 24 and 48 hours after removal of the dressing. Under the conditions of the challenge phase (tested concentrations: 100%, 50%, 25%, 10%, 5% and 1% (w/w)): the filter paper of a chamber (Finn Chamber) was fully-loaded with a dosage form preparation. The chamber was then applied to the clipped area of the skin (one concentration per flank). The chamber was held in place by means of an occlusive dressing for 24 hours, cutaneous reactions were evaluated 24 and 48 hours after removal of the dressings.

Main test:
Intradermal route induction: On day 1, six injections were made deep into the dermis of a 4 cm x 2 cm clipped interscapular area, using a needle (diameter: 0.50 x 16 mm) mounted on a 1 ml plastic syringe (0.01 ml graduations). Three injections of 0.1 ml were made into each side of this interscapular region (i.e. three pairs of sites; Table 1). The anterior and middle pairs of injections were performed close to each other and nearest the head, while the posterior pair was performed towards the caudal part of the test area.
Cutaneous route induction: As the test item was shown to be irritant during the preliminary test, a topical application of sodium lauryl sulfate was not necessary on day 7. On day 8, a pad of filter paper (approximately 8 cm2) was fully-loaded with the test item at the concentration of 10% (w/w) and was then applied to the interscapular region of the animals of the treated group. The animals of the control group received an application of the vehicle alone under the same experimental conditions. The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster. On removal of the dressing (day 10), no residual test item was observed. A local irritation was recorded in all the animals of the control and treated groups.
First challenge application: On day 22, the animals of treated and control groups received an application of the test item and vehicle. The filter paper of a chamber (Finn Chamber) was fully-loaded with the test item at the concentration of 5% (w/w) and was then applied to a clipped area of the skin of the posterior right flank of all animals. The vehicle was applied under the same experimental conditions to the skin of the posterior left flank. The chambers were held in contact with the skin for 24 hours by means of an adhesive anallergenic waterproof plaster. As equivocal cutaneous reactions were noted, a second challenge application was performed after a rest period of 13 days.
Second challenge application: On day 36, the animals of treated and control groups received an application of the test item at the concentration of 1% (w/w) to the median left flank and the vehicle to the median right flank, under the same experimental conditions as for the first challenge application.

The animals were observed at least once a day to check for clinical signs and mortality. Individual body weights were recorded on the day of group allocation, the first study day (day 1), on day 25 and day 39.
Challenge controls:
See Table 1.
Positive control results:
Under our experimental conditions and according to the Magnusson and Kligman method, the test item Mercaptobenzothiazole induced positive skin sensitization reactions in 100% (10/10) guinea pigs.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
5% (w/w)
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5% (w/w)
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
in addition, one animal that scored negative showed 'dryness of skin'.
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
Mercaptobenzothiazole 20% (w/w)
No. with + reactions:
10
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
Mercaptobenzothiazole 20% (w/w)
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5% (w/w)
No. with + reactions:
12
Total no. in group:
19
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5% (w/w)
No. with + reactions:
14
Total no. in group:
19
Clinical observations:
In one animal scoring was masked by dryness of the skin
Remarks on result:
not determinable
Remarks:
In order to determine whether the observed cutaneous reactions are attributable to delayed contact hypersensitivity or to an irritant effect of the test item, a second challenge application was performed. For this second challenge application, the lower concentration of 1% (w/w) was chosen.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
1% (w/w)
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
1% (w/w)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
1% (w/w)
No. with + reactions:
12
Total no. in group:
19
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
1% (w/w)
No. with + reactions:
9
Total no. in group:
19
Remarks on result:
positive indication of skin sensitisation

Preliminary test: In order to respect the criteria for the selection of concentrations (the concentrations should be well-tolerated systemically and locally, cutaneous application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration, cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effect), the concentration chosen for the topical application of the induction phase (day 8) was 10% (w/w). For the challenge application (day 22), it was 5% (w/w).

Main test: One animal of the treated group was found dead on day 2; no clinical signs were observed prior to death. As such spontaneous mortality is sometimes observed in this species, it was not attributed to treatment with the test item. No clinical signs and no deaths related to treatment were observed during the study. The body weight gain of the treated animals was similar to that of controls.

First challenge application: A discrete erythema (grade 1) was observed in 2/10 animals of the control group at the 48-hour reading. Dryness of the skin was recorded in 1/10 animals at the 48-hour reading. In the treated group, at the 24-hour reading, a discrete or moderate erythema (grade 1 or 2), together with crusts in one animal, was noted in 10/19 and 2/19 animals, respectively. At the 48-hour reading, a discrete or moderate erythema (grade 1 or 2) was recorded in 6/19 and 8/19 animals, respectively. Dryness of the skin, which masked the evaluation of the erythema in one animal, was observed at the 48-hour reading in 14/19 animals. Crusts in one animal and oedema in another one were also noted. In order to determine whether the observed cutaneous reactions are attributable to delayed contact hypersensitivity or to an irritant effect of the test item, a second challenge application was performed. For this second challenge application, the lower concentration of 1% (w/w) was chosen.

Second challenge application: No cutaneous reactions were recorded in the animals of the control group. In the treated group, at the 24-hour reading, a discrete or moderate erythema (grade 1 or 2) was observed in 11/19 and 1/19 animals, respectively. At the 48-hour reading, a discrete or moderate erythema (grade 1 or 2), together with an oedema and/or dryness of the skin in four animals, was noted in 6/19 and 3/19 animals, respectively. The persistent cutaneous reactions observed in 9/19 animals of the treated group after the second challenge application were attributed to delayed contact hypersensitivity.

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
According to the classification criteria laid down in Council Directive 67/548/EEC (and subsequent adaptations) on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labeling of dangerous substances, the test item CECABASE 280 should be classified as sensitizing to the skin and assigned the symbol Xi, the indication of danger "Irritant" and the risk phrase R 43: "May cause sensitisation by skin contact".
Executive summary:

The potential of CECABASE 280 (CAS: 71820 -35 -4) to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximisation method of Magnusson and Kligman.

On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals: Freund's complete adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl (both groups); test item at the concentration of 1% in corn oil (treated group) or vehicle alone (control group); test item at the concentration of 1% in a mixture FCA/0.9% NaCl (50/50, w/w) (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl (50/50, v/v) (control group). On day 8, the animals of the treated group received a topical application of the test item at the concentration of 10% (w/w) in ethanol/water (80/20) to the same test site, which was then covered by an occlusive dressing for 48 hours. The animals of the control group received an application of the vehicle under the same experimental conditions. On day 22, all animals of both groups were challenged by a cutaneous application of the test item at the concentration of 5% (w/w) in acetone to the right flank. The test item was maintained under an occlusive dressing for 24 hours. The vehicle was applied to the left flank under the same experimental conditions. Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing. As equivocal cutaneous reactions were noted after the first challenge, a second challenge application was performed on day 36. The test item at the concentration of 1% (w/w) in acetone was applied to the left flank and the vehicle to the right flank of the animals of both groups, under the same experimental conditions as for the first challenge application.

No clinical signs and no deaths related to treatment were noted during the study. After the first challenge application, a discrete erythema was observed in 2/10 animals of the control group at the 48-hour reading. Dryness of the skin was recorded in an additional animal at the 48-hour reading. In the treated group, at the 24-hour reading, a discrete or moderate erythema, together with crusts in one animal, was noted in 10/19 and 2/19 animals, respectively. At the 48-hour reading, a discrete or moderate erythema was recorded in 6/19 and 8/19 animals, respectively. Dryness of the skin, which masked the evaluation of the erythema in one animal, was observed at the 48-hour reading in 14/19 animals. Crusts in one animal and oedema in another one were also noted.

After the second challenge application, no cutaneous reactions were recorded in the animals of the control group. In the treated group, at the 24-hour reading, a discrete or moderate erythema was observed in 11/19 and 1/19 animals, respectively. At the 48-hour reading, a discrete or moderate erythema, together with an oedema and/or dryness of the skin in four animals, was noted in 6/19 and 3/19 animals, respectively. The persistent cutaneous reactions observed in 9/19 animals of the treated group after the second challenge application were attributed to delayed contact hypersensitivity.

Under the conditions of the study, and according to the maximisation method of Magnusson and Kligman, the test substance induced delayed contact hypersensitivity in 9/19 (47%) guinea pigs and should therefore be considered as a moderate skin sensitiser.

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

Two Guinea Pig maximisation studies are available. The studies were conducted in accordance with the OECD Guideline 406 (Skin Sensitisation). The objective of these studies was to evaluate the potential of the test item to induce skin hypersensitivity in guinea pigs following intradermal and epidermal exposure of the test animals to the respective test substances.

 

The study conducted by Ollivier (2004) investigated the potential of CECA BASE 280 to induce delayed contact hypersensitivity in guinea pigs. 30 Hartley guinea Pigs (10 male and 10 female in the treated groups and 5 per sex in the control group) received 3 pairs of intradermal injections with 1% test substance concentration into the interscapular region of all animals. One animal died at day 2 of the study, which was not considered test substance related. 8 Days after intradermal induction, the test animals received a topical application 10% of the test item (in EtOHwater 80/20 w/w%) to the same test site which was then covered with an occlusive dressing for 48 hours. On day 22, all animals were exposed to a challenge by cutaneous application of 5% test substance in Acetone to the right flank of the test animals for 24 hours.In total, persistent (both at 24 and 48 hour) cutaneous reactions were observed in 12/19 (63%) animals of the treated group. However, due todiscrete erythema observed at 48 hoursin control animals, a re-challenge with 1% in Acetone was performed. At this challenge concentration 12/19 (63%) animals showed dermal reactions at 24 hr, whereas none of the control animals showed a reaction. At 48 hr reading 9/19 (47%) showed cutaneous reactions.

Based on the results of this study, CECA BASE 280 should be considered to be a skin sensitizer.

 

In the study conducted by Pels Rijcken (1995) the ability of Tall oil reaction products with aminoethylpiperazine to induce skin sensitization in the Albino Guinea Pig was investigated. 10 test animals were intradermally injected with a 0.5% concentration and epidermally exposed to a 1% test concentration. 2 weeks following application, the animals were challenged with a 1%, 0.5% and 0.1% test substance concentration. One animal removed bandages and was excluded from evaluations. At 1% all test animals but also 3/5 control animals showed dermal responses indicating that this level was too high. At 0.5% all 9/9 test animals (100%) showed dermal responses and at 0.1% 3/9 (33%) showed reactions, whereas none of the control animals showed dermal responses.

The results of this study indicate that the substance should be considered to be a skin sensitiser.

 

Overview of the two studies:

Parameter

Pels Rijcken (1995)

Ollivier (2004)

Animals test/control

9/5 (one test animal lost for evaluation)

19/10 (one test animal died)

Induction – i.d.

0.5% in corn oil

1% in corn oil

Induction - epidermal

0.5 ml 1% in corn oil covered by 2x4 cm patch.

8cm2 filter paper fully loaded with 10% in EtOH/water (80/20)

Challenge

0.05 ml 0.1%, 0.5%, 1% in corn oil using square chambers.

fully loaded Finn Chamber with 5% in Acetone

Re-challenge 1%

Response test - control

0.1%: 3/9 - 0/5 (33%)

0.5%: 9/9 - 0/5 (100%)

1% : 9/9 - 3/5 (50% test animals more severe reactions than the maximum of control)

5%: 15/19 - 2/10; (dryness at 48 hr masking reading)

1%: 24h: 12/19 - 0/10 (63%);

48h: 9/19 - 0/10 (47%, 3 test animal recovered)

 

For corrosive substances, the use of protective gloves and other equipment, such as face shields, aprons and good work practices are mandatory. As a result, direct dermal contact is very unlikely.Lack of exposures explains that although the substance can be considered a strong sensitiser based on the results from both studies, the producers are not aware of reported incidences of sensitisation among own employees or from customers.

Migrated from Short description of key information:

The substance was found to be sensitizing in two different Guinea pig maximization studies.

Justification for selection of skin sensitisation endpoint:

Considered the most valid study of the two available studies mostly related to limited information reported about the tested substance in the study of Pels Rijcken (1995).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Justification for selection of respiratory sensitisation endpoint:

Tall oil reaction products with aminoethylpiperazine has a low vp pressure (<3 x 10-7 Pa at 23°C) and its use is limited to industrial and professional settings that do not involve the forming of aerosols, particles or droplets of an inhalable size. So exposure to humans via the inhalation route will be unlikely to occur.

Justification for classification or non-classification

Skin Sensitisation:

According to the results of the study by Ollivier (2004), the test substance should be considered to be a skin sensitiser, due to the persistent (positive reaction at both 24 and 48 hour reading) cutaneous reactions observed in 63% of the treated test animals observed following the first challenge with 5%.As equivocal cutaneous reactions were noted after the first challenge, a second challenge application was performed with 1%. This resulted to apersistent cutaneous reactions observed in 9/19 (47%) animals of the treated group.

 

The study fromPels Rijcken (1995) resulted to a 100% (9/9) sensitisation in animals challenged with 0,5% and following induction of 0.5% intradermally and 1% epidermally.

 

According to CLP criteria (ATP 2 - Commission Regulation 286/2011 of 10 March 2011) a substance needs to be classified as skin sensitizer based on the results of a Guinea pig maximization test:

-      30% of the animals show a positive reaction and where the intradermal induction was performed applying concentrations 0.1%, or 60% responding following > 0.1% to 1% intradermal induction dose.

-      30% responds following > 1% intradermal dose, or 30% to < 60% responds following >0.1% to 1% intradermal induction dose.

 

Ollivier (2004): 47%-63% positive following 1% i.d. concentration => Equivocal between 1A and 1B

Pels Rijcken (1995): 100% positive following 0.5% i.d. concentration => Cat. 1A

 

In conclusion, based on available dataTall oil reaction products with aminoethylpiperazineshould beclassified as Skin sensitizer Category 1A, with hazard statement, H317: May cause an allergic skin reaction.

 

Respiratory sensitisation:

There is no data available with respect to respiratory sensitization byTall oil reaction products with aminoethylpiperazine. However, followinga very low vp pressure (<3 x 10-7 Pa at 23°C) anduse limited to industrial and professional settings that do not involve the forming of aerosols, particles or droplets of an inhalable size, exposure of humans via the inhalation route will be unlikely.