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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:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Remarks:
Not specified in report
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
and "Allergic Contact Dermatitis in the Guinea-Pig: Identification of Contact Allergens" Magnusson B. Kligman A.M., 1970 published by C.C. Thomas, Springfield, Illinois, USA.
Deviations:
no
Remarks:
Not specified in report.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study performed in 1998 prior to the implementation of the REACH regulation and the need to perform skin sensitising studies using either the LLNA or in vitro/in chemico assays.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
Species: Dunkin Hartley strain, albino guinea pig (SPF-quality) Recognised by international guidelines as the recommended test system (e.g. OECD, EC).
Source : Charles River, Germany.
Number of animals Experimental group : 10 females, Control group : 5 females. (females were nulliparous and non-pregnant).
Age and body weight: Young adult animals (approx. 5 weeks old) were selected. Individual body weights did not exceed 500 grams.
Identification: Ear tattoo.
Reliability check: The results of a reliability test performed not more than 6 months previously. Similar procedures were used in the reliability test and
in this study.

Conditions
Air-conditioned room with approximately 15 air changes per hour and the environment controlled with optimal conditions considered as being a temperature of 21°C and a relative humidity of 50%. Fluctuations from these optimal conditions were noted, but were considered not to have affected study integrity. Lighting was 12 hours artificial fluorescent light and 12 hours dark per day.

Accommodation
Group housing of 5 animals per labelled metal cage with wire-mesh floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands). The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions.

Diet
Free access to standard guinea pig diet , including ascorbic acid (1600 mglkg); LC 23-B, pellet diameter 4mm (Hope farms, Woerden, The Netherlands). Certificate of analysis were examined and retained i n the NOTOX archives. In addi i o n , hay (B.M.I., Helmond, The Netherlands) was provided once a week.

Water
Free aecess to tap-water, diluted with decalcefied water. Certificates of quaterly analysis for tap-water were examined and retained in the NOTOX archives.
Route:
other: intradermal and epidermal occlusive
Vehicle:
other: Water (Milli-U)
Concentration / amount:
0.5% a.i. concentration for the intradermal induction and 50% a.i. concentration for the epidermal induction exposure
20 and 50% a.i. 1st challenge
5 and 10% a.i. 2nd challenge
Route:
epicutaneous, occlusive
Vehicle:
other: Water (Milli-U)
Concentration / amount:
0.5% a.i. concentration for the intradermal induction and 50% a.i. concentration for the epidermal induction exposure
20 and 50% a.i. 1st challenge
5 and 10% a.i. 2nd challenge
No. of animals per dose:
10
Details on study design:
Preliminary Irritation Study
Prior to the start of the main study, the intradermal and epidermal irritancy of DOWFAX 8390-D was investigated to select test substance concentrations suitable for the induction and challenge phase of the main study. The selection was done in consultation with the sponsor and was based on the absence of toxicity and on the following criteria for each route and/or study phase:

Induction (intradermal and epidermal):
The highest possible concentration that produced moderate irrittion (the intradermal reactions may include slight necrosis(< 3 mm in diameter)).

Challenge:
The maximum non-irritant concentrations. Selection of this concentration depended on a number of factors and exact criteria did not always apply.

Results:
The test substance concentrations selected for the main study were 0.5% concentration for the intradermal induction and 50% a.i. concentration for the epidermal induction exposure. The test site of all animals was treated with 10% SDS approximately 24 hours before the epidermal induction in the main study, to provide a mild inflammatory reaction. A 50% and 20 % a.i. test substance concentration were selected for the challenge phase.

INDUCTION - Experimental animals
Day 1
The scapular region was clipped and three pairs of intradermal injections (0.1 ml/site) were made in this area as follows :
A) A 1:1 wlw mixture of Freunds' Complete Adjuvant ( Difco, Detroit, U.S.A.) with water for injection (Fresenius AG, Bad Homburg, Germany).
B) The test substance at a 0.5% concentration.
C) A 1:l wlw mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant. Note: One of each pair was on each side of the midline and from cranial A) to caudal C ). Day 3 The dermal reactions caused by the intradermal injections were assessed for irritation.

Day 7
The scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate (SDS, Boom, Meppel, The Netherlands) i n vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.

Day 8
The 10% SDS treated area between the injection sites was treated with 0.5 ml of a 50% test substance concentration using a Metalline patch (2x3 cm)
mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.
The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance and the dermal reactions caused by the epidermal
exposure were assessed for irritation.

INDUCTION - Control animals
The control animals were treated as described for the experimental animals, except that, instead of the test substance, the vehicle was administered.

CHALLENGE - All animals
Day 22
One flank of all animals was clipped and treated by epidermal application of a 50% and a 20% test substance concentration and the vehicle (0.5 rnl
each), using Metalline patches (2x3 cm) mounted on Medical tape, which were held in place with Micropore tape and subsequently Coban elastic
bandage.

The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle. The treated sites were assessed for
challenge reactions 24 and 48 hours after removal of the dressing.

Day 29
A re-challenge was conducted approximately one week after the first challenge, to clarify the results in the first challenge. The contralateral flank of all animals was similarly treated, except that a 10% and a 5% test substance concentration and the vehicle were used.

HISTOPATHOLOGY
Of all animals the test and vehicle treated sites (second challenge phase only) were excised, fixed in a neutral phosphate buffered 4% formaldehyde solution and stored in labelled containers. No histopathological examination was performed.

OBSERVATIONS
Mortality/Viability: Twice daily
Toxicity: At least once d a i l y .
Body weights: Prior to start and at termination of the study.
Necropsy:The animal removed from the study was subjected to necropsy for gross macroscopic examination.
Irritation: Skin reactions were graded according to the following numerical scoring systems. Furthermore, a description of all other (local )effects was recorded. Whenever necessary, the treated skin-areas were clipped at least 3 hours before the next skin reading to facilitate scoring.

Grading Irritation Reactions:
Erythema and eschar formation:
No erythema .............................................................................0
Slight erythema (barely perceptible) .................................................... 1
Well-defined erythema ................................................................... 2
Moderate erythema ....................................................................... 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) ........... 4

Oedema formation:
No oedema ............................................................................... 0
Slight oedema (barely perceptible) ...................................................... 1
Well-defined oedema (edges of area well-defined by definite raising) . . . . . . . . . . . . . . . . . . . 2
Moderate oedema (raised approximately 1 millimeter) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) . 4

Intradermal reactions were assessed for erythema only or, if necrosis is present, the diameter of necrosis.

Grading Challenge Reactions:
No visible change ........................................ 0
Discrete or patchy erythema ............................. 1
Moderate and confluent erythema ......................... 2
Moderate erythema and swelling .......................... 3
Intense erythema and swelling ........................... 4
After the end of the study all animals were killed by asphyxiation using an oxygen/carbon dioxide procedure.
Challenge controls:
5
Positive control substance(s):
yes
Remarks:
Alpha-Hexylcinnamic Aldehyde, Tech. 85%
Vehicle:
other: Not applicable
Concentration:
Not applicable
No. of animals per dose:
Not applicable
Details on study design:
Not applicable
Statistics:
None
Positive control results:
The skin reactions in the experimental animals observed in response to the 10% and 5% of positive control substance in the challenge phase were considered indicative of sensitization, based on the absence of any response in the control animals.

Exposure to postive control lead to a sensitization rate of 100 per cent to both the 10% and 5% concentrations. From these results, it was concluded that the female guinea pig of the albino Dunkin Hartley strain is an appropriate animal model for the performance of studies designed to evaluate the sensitizing potential of a substance in a Maximisation type of test.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
10% a.i. of test material
No. with + reactions:
1
Total no. in group:
9
Clinical observations:
One animal was removed from study due to ill health unrelated to treatment.
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 10% a.i. of test material. No with. + reactions: 1.0. Total no. in groups: 9.0. Clinical observations: One animal was removed from study due to ill health unrelated to treatment..
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
5% a.i. of test material
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 5% a.i. of test material. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
10% a.i. of test substance
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
None
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 10% a.i. of test substance. No with. + reactions: 2.0. Total no. in groups: 5.0. Clinical observations: None.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
5% a.i. of test material
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: 5% a.i. of test material. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
10% a.i. of test material
No. with + reactions:
1
Total no. in group:
9
Clinical observations:
One animal was removed from study due to ill health unrelated to treatment. Scaliness was observed in one animal.
Remarks on result:
other: see Remark
Remarks:
Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 10% a.i. of test material. No with. + reactions: 1.0. Total no. in groups: 9.0. Clinical observations: One animal was removed from study due to ill health unrelated to treatment. Scaliness was observed in one animal..
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
5% a.i. of test material
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 5% a.i. of test material. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
9
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 9.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
10% a.i. of test material
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 10% a.i. of test material. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
5% a.i. of test material
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 5% a.i. of test material. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: vehicle. No with. + reactions: 0.0. Total no. in groups: 5.0.
Parameter:
SI
Remarks on result:
other: Not applicable
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not applicable

MAIN STUDY

Induction phase

The reactions noted in the experimental and control animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

Challenge phase

First Challenge

Skin reactions varying between grades 1 and 2 were observed in eight experimental and three control animals in response to the 50% a.i. test substance concentration and similar reactions were noted in five out of 9 experimental animals and two out of 5 control animals in response to the 20% a.i. concentration. Scaliness was seen in some treated skin sites among the experimental and control animals.

Second challenge

Since skin reactions were observed in both experimental and control animals in first challenge, a second challenge was performed one week later. The animals were then topically treated with the 10% and 5% a.i. test substance concentration.

Skin reactions of grade 1 were observed in one out of 9 experimental and two out of 5 control animals in response to the 10% a.i. test substance concentration.

No skin reactions were observed in any of animals on the study in response to the 5% a.i. concentration. Scaliness was seen in the treated skin sites of one experimental animal.

Toxicity/Mortality

One experimental animal (no 6) was removed from the study after showing signs of ill health (quick breathing, laboured respiration, dark eyes, pale skin,

piloerection ). Macroscopic examination of this animal showed emaciation, enlarged spleen, dark red discolouration of the lungs and gray white discoloured patches in the cortex of the kidneys. It was considered that the signs of ill health shown by this animal were incidental and that the study outcome, based on the healthy surviving animals, was not adversely affected. No further mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Body Weights

Slightly reduced body weight gain was noted in one experimental animal (no. 9 ). This event was considered not to have affected the study, since the skin reactions

noted in this animal were similar to the other animals. Body weights and body weight gain of other experimental animals remained in the same range as controls over the study period.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In response to the 50%, 20% and 10% a.i. concentration, comparable irritation reactions were noted in control and experimental animals to the same concentrations. Moreover, the number of animals that showed skin reactions decreased at lower concentrations, resulting in no reactions in any of the animals in response to the 5% a.i. concentration. It was therefore considered that the skin reactions observed were indicative of non-specific irritation and that no hypersensitivity had been induced in test animals.

These results indicate a sensitization rate of 0%.

Thus, DOWFAX* 8390-D is not a skin sensitizer and does not have to be classified according to the criteria of the Commission of the European Communities (Commission Directive 93/21/EEC).
Executive summary:

A study was performed to assess the skin contact sensitization potential of DOWFAX* 8390-D in the albino guinea pig. The study conformed to the OECD Guidelines for Testing of Chemicals No. 406 and to Commission Directive 96/54, Annex V, Part B; B.6: skin sensitization.

The concentration of the test material used in the main study was based on the results of a pretest. Ten test and five control female Dunkin-Hartley guinea pigs were used for the main study. Intradermal induction consisted of two injections (0.1 ml per site) of the test material (0.5% a.i. w/v in water), with and without Freund's Complete Adjuvant, and a control with Adjuvant alone. After one week, the scapular area between the two intradermal injection sites (one day before the topical treatment the skin was rubbed with 10 % (w/v) sodium dodecyl sulfate toincrease sensitivity) was treated topically for 48 hours with 0.5 ml of a 50% a.i. (w/v) dilution of the test material in water. Induction readings were made at day 3 (after intradermal injection) and day 10 (after epidermal exposure). Challenge on day 22 consisted of a single, 24-hour, topical application (0.5 ml) of the test material at concentrations of 20% and 50% a.i. w/v in water on two separate sites on the right flank of each test and control animal under an occlusive dressing. Observations for any dermal reaction were made approximately 24 and 48 hours after removal of the dressing. A rechallenge was conducted approximately one week after the first challenge with 5% and 10 % a.i. (w/v) of the test material.

During the induction phase, severe erythema was noted at all intradermal injections sites treated with Adjuvant and test material including signs of necrosis with three animals. Well defined to moderate erythema was observed in most cases of the epidermally treated induction sites of the experimental animals. Between the controls and the experimental animals there was no significant difference in skin reactivity noted at the challenge sites 24 and 48 hours after removal of the dressings with corresponding test material concentrations of 5, 10, 20, and 50% a.i.

Thus, DOWFAX* 8390-D is not a skin sensitizer and does not have to be classified according to the criteria of the Commission of the European Communities (Commission Directive 93/21/EEC).

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

A number of sensitization studies with the Dowfax 8390 material were conducted over a range of active ingredient (a.i.) concentrations. General findings included non-specific skin irritation during induction applications and in challenged naïve and test animals at high a.i. concentrations. In all but one test, where Dowfax 8390 concentrations tested were above 7% a.i., skin reactions in guinea pigs were observed, both in test and naïve control groups. In addition, in the two equivocal studies the appropriate controls or procedures were not conducted.

In the key GPMT study, significant proportion of test and naïve control animals reacted to 50% and 20% a.i. challenge concentrations of Dowfax 8390. The animals were subsequently re-challenged with lower Dowfax 8390 concentrations of 10% and 5% a.i. While 10% a.i. induced responses in guinea pigs, Dowfax 8390 at 5% a.i. did not induce skin reactions in test and naïve control animals. The results of this well conducted key study indicate that Dowfax 8390 is not a skin sensitizer. 

Moreover, no skin reactions were observed in animals challenged with topical Dowfax 8390 treatments of 2% and 0.5% a.i. concentrations. Together, the weight of evidence suggests that Dowfax 8390 surfactant is a skin irritant, and stimulates skin irritation responses in animals but is not a skin sensitizer.


Short description of key information:
General findings from a number of sensitization studies with Dowfax 8390 included non-specific irritation during induction applications and in challenged naïve and test animals at high active ingredient (a.i.) concentrations. In the key GPMT study, significant proportion of test and naïve control animals reacted to 50% and 20% a.i. challenge concentrations of Dowfax 8390. The animals were subsequently re-challenged with lower Dowfax 8390 concentrations of 10% and 5% a.i. While 10% a.i. induced responses in tested guinea pigs, Dowfax 8390 at 5% a.i. did not induce skin reactions in test animals or naïve control animals. The results of this well conducted key study indicate that Dowfax 8390 is not a skin sensitizer.

Justification for selection of skin sensitisation endpoint:
Well conducted sensitization test with naive control group included and challenge performed down to the approprite concentration that did not induce reactions in naive control animals.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

General findings with Dowfax 8390 point to non-specific skin irritation responses in the two equivocal studies where appropriate controls or procedures were not conducted. The key study and weight of evidence (WoE) from all of the supporting studies indicate that Dowfax 8390 is not a skin sensitizer. Therefore, classification concerning skin sensitization according to CLP GHS and the Directive 67/548/EEC (DSD) is not warranted for the substance.

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