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Description of key information

Skin Sensitization:

The dermal sensitization potential of test chemical was evaluated in a mouse local lymphnode assay(LLNA). The study was performed as per OECD 429 Guidelines.

The relative potency index of test chemical was calculated to be 21.8.

Based on the relative potency index, test chemical was considered to be a weak sensitizer.

The skin sensitization effects were observed for test chemical in human subjects (male and female) according to Repeated insult patch test.

Under the conditions of this test none of the 37 subjects tested was sensitized by the test sample.

Therefore the test chemical was considered as non- skin sensitizing to humans.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
data is from peer reviewed journals
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
To evaluate the potential of test chemical for inducing allergic contact dermatitis
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7-12 weeks
Vehicle:
other: ethanol:diethyl phthalate
Concentration:
25µl of the test material in ethanol:diethyl phthalate
No. of animals per dose:
Groups of female CBA mice (7-12 weeks of age)
Details on study design:
MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
A substance was classified as a skin sensitizer if it induced a threefold or greater increase in local lymph node proliferative activity at one or more test concentrations when compared with concurrent vehicle-treated controls (SI≥3). Dose response data were used to measure the relative skin sensitization potency of all of the chemicals that were positive. When the LLNA dose-response curve included concentrations that induced at least one SI greater than 3 and one SI less than 3, EC3 values were calculated by linear interpolation. For chemicals that induced an SI greater than or equal to 3 at all concentrations tested, an EC3 value was extrapolated from the two lowest doses used. For this extrapolation method to work, a dose response should be evident. The relative sensitizing potencies of the chemical allergens were categorized via a recently proposed arbitrary classification scheme.

TREATMENT PREPARATION AND ADMINISTRATION: groups of CBA female mice (7–12 weeks of age) were exposed topically on the dorsum of both ears to 25 µL of test material or to an equal volume of the relevant vehicle alone. Treatment was performed daily for 3 consecutive days. Five days after the initiation of exposure, all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of tritiated thymidine. Mice were sacrificed 5 hours later, and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by beta scintillation counting was reported in disintegrations per minute (dpm). A stimulation index (SI) was calculated for each chemical-treated group as the ratio of the dpm of the treated group (or mean dpm when individual animals were assessed) to the dpm or mean dpm of the concurrent vehicle control group.
Positive control substance(s):
not specified
Parameter:
EC3
Value:
21.8
Test group / Remarks:
test group
Remarks on result:
other: positive sensitizer

Table: Chemical Structures, Molecular Weights, LLNA Data, Potency Categorizations, and Reaction Mechanistic Domains

CAS

Vehicle

LLNA%

LLNA%

LLNA%

LLNA%

LLNA%

LLNA SI

LLNA SI

LLNA SI

LLNA SI

LLNA SI

LLNA EC3

Relative Potency

Reaction mechanism domain

6485-40-1

E:D

2.5

5.0

10.0

25.0

50.0

0.6

0.6

1.5

3.4

4.6

21.8

Weak

MA

E:D –ethanol:diethyl phthalate; LLNA – Local lymph node assay (LLNA% = weight per volume concentration); EC3 – Mathematically estimated concentration of the test chemical necessary to induce a threefold stimulation index; MA –Michaels acceptor

**value is estimated

Interpretation of results:
other: sensitizing
Conclusions:
The relative potency index of test chemical was calculated to be 21.8.
Based on the relative potency index, test chemical was considered to be a weak sensitizer.
Executive summary:

The dermal sensitization potential of test chemical was evaluated in a mouse local lymphnode assay(LLNA). The study was performed as per OECD 429 Guidelines. Groups of female CBA mice (7-12 weeks of age) were exposed topically on the dorsum of both ears to 25µl of the test material in ethanol:diethyl phthalate or to an equal volume of ethanol:diethyl phthalate only. Treatment was performed daily for 3 consecutive days.Five days after the initiation of exposure, all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of tritiated thymidine. Mice were sacrificed 5 hours later, and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by beta scintillation counting was reported in disintegrations per minute (dpm). A stimulation index (SI) was calculated for each chemical-treated group as the ratio of the dpm of the treated group (or mean dpm when individual animals were assessed) to the dpm or mean dpm of the concurrent vehicle control group.The approach to estimation of the relative skin sensitization potential was based on the mathematical estimation of the concentration of chemical necessary to obtain a threshold positive response (SI = 3); this is termed as the EC3 value.A substance was classified as a skin sensitizer if it induced a threefold or greater increase in local lymph node proliferative activity at one or more test concentrations when compared with concurrent vehicle-treated controls (SI≥3).

The relative potency index of test chemical was calculated to be 21.8.

Based on the relative potency index, test chemical was considered to be weak sensitizer.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Justification for type of information:
data from experimental report following standard testing procedure.
Qualifier:
according to guideline
Guideline:
other: Repeated insult patch test
Principles of method if other than guideline:
The skin sensitization effects were observed for test chemical in human subjects (male and female) according to Repeated insult patch test.
GLP compliance:
not specified
Type of study:
patch test
Justification for non-LLNA method:
not specified
Species:
human
Sex:
male/female
Details on test animals and environmental conditions:
subjects were furnished by churches, parent- teachers associations and similar organizations in suburban areas arount Cincinnati.
Route:
epicutaneous, occlusive
Vehicle:
no data
Concentration / amount:
0.5 ml (12.5%)
Day(s)/duration:
24 hrs
Adequacy of induction:
not specified
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
no data
Concentration / amount:
0.5 ml (12.5%)
Day(s)/duration:
24 hrs
Adequacy of challenge:
not specified
No. of animals per dose:
The test was initiated on 43 subjects, of whom 37 completed the program.
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 9
- Exposure period: 24 hours
- Test groups:37
- Control group: no data
- Site: on the subject’s upper arm
- Frequency of applications: nine 24 hours exposure on a Monday-Wednesday- Friday for 3 successive weeks
- Duration: 3 weeks
- Concentrations:0.5mL (12.5 %)

B. CHALLENGE EXPOSURE
- No. of exposures:1
- Day(s) of challenge:14 Days (on Monday of sixth week)
- Exposure period: 24 hours
- Test groups: 37
- Control group: no data
- Site: patch was applied to a site not previously exposed
- Concentrations: 0.5mL (12.5 %)
- Evaluation (hr after challenge): On Wednesday or Friday of 6th week. (after 24 and 72 hours of challenge exposure )
Challenge controls:
no data
Positive control substance(s):
not specified
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0.5mL (12.5 %)
No. with + reactions:
0
Total no. in group:
37
Clinical observations:
Skin sensitization was not observed.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
0.5mL(12.5 %)
No. with + reactions:
0
Total no. in group:
37
Clinical observations:
Skin sensitization was not observed.
Remarks on result:
no indication of skin sensitisation

Initially ten subjects were started on test sample. If no outoward results were observed upon completion of the test with these, enough additional subjects were started to bring the total to about 40 subjects. occasional subjects failed to complete the test for various reasons irrelevant to the purpose of the study, such as loss of interest, nausea or respiratory distress attributed to sample odors, unexpected other obligations and so on.

Interpretation of results:
other: not sensitising
Conclusions:
Under the conditions of this test none of the 37 subjects tested was sensitized by the test sample.
 
Therefore the test chemical was considered as non- skin sensitizing to humans.
Executive summary:

The skin sensitization effects were observed for test chemical in human subjects (male and female) according to Repeated insult patch test.

 

This test procedure was an adaptation of the repeated insult patch test procedure suggested by Draize.The test was initiated on 43 subjects, of whom 37 completed the program.

0.5ml of test sample was applied 9 times on upper arm of each subject for 3 successive weeks (on a Monday-Wednesday- Friday) for exposure period of 24 hours. After 14 days of induction on Monday of sixth week challenge patch was applied to a site not previously exposed and removed after 24 hours and the reactions were scored on Wednesday and Friday of this week. Under the conditions of this test none of the 37 subjects tested was sensitized by the test sample.

 

Therefore the test chemical was considered as non- skin sensitizing to humans.

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

Skin Sensitization

Various studies have been summarized to determine allergenic potential caused by the test chemical in living organisms. These results include in vivo experimental data in humans as well as mice. The results are summarized below:

The skin sensitization effects were observed for test chemical in human subjects (male and female) according to Repeated insult patch test.

This test procedure was an adaptation of the repeated insult patch test procedure suggested by Draize. The test was initiated on 43 subjects, of whom 37 completed the program.

0.5ml of test sample was applied 9 times on upper arm of each subject for 3 successive weeks (on a Monday-Wednesday- Friday) for exposure period of 24 hours. After 14 days of induction on Monday of sixth week challenge patch was applied to a site not previously exposed and removed after 24 hours and the reactions were scored on Wednesday and Friday of this week. Under the conditions of this test none of the 37 subjects tested was sensitized by the test sample.

Therefore the test chemical was considered as non- skin sensitizing to humans.

This is supported by the results of a patch test conducted on 28 human subjects. All the volunteers were selected on a random basis, except that no one was accepted for the study that had active dermatologic conditions or other active illness. The sample patch test was performed on 5 volunteers as a preliminary check for irritation. Since no reactions were found in this 5 people after 24 hours, the remaining 23 individual also had their arms patched. Thus the pilot group of 5 individual received 11 sensitizing patch tests while the remaining 23 subjects received 10 patch applications in sensitization series.

During induction, 0.5 ml (10%) of sample was applied on arms of each subject as closed patches which were subsequently secured by overlying strips of firmly adhesive tape. The patches were placed upon the skin or removed on Mondays, Wednesdays and Fridays. 2 weeks later challenge was performed after the last patch application of induction and the skin reactions were observed at 48 and 72 hours.

Out of 28 subjects, only one 29 year old female did not report for the challenge test. Thus 27 people responded to the challenge dose and the test gave no indication of sensitization. Since volunteers did not induce any sensitization reaction, the test chemical was considered as non- skin sensitizing to humans subjects.

The dermal sensitization potential of test chemical was also evaluated in a mouse local lymphnode assay(LLNA). The study was performed as per OECD 429 Guidelines. Groups of female CBA mice (7-12 weeks of age) were exposed topically on the dorsum of both ears to 25µl of the test material in ethanol:diethyl phthalate or to an equal volume of ethanol:diethyl phthalate only. Treatment was performed daily for 3 consecutive days.Five days after the initiation of exposure, all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 20 µCi of tritiated thymidine. Mice were sacrificed 5 hours later, and the draining auricular lymph nodes were excised and pooled for each experimental group or each individual animal. The incorporation of tritiated thymidine measured by beta scintillation counting was reported in disintegrations per minute (dpm). A stimulation index (SI) was calculated for each chemical-treated group as the ratio of the dpm of the treated group (or mean dpm when individual animals were assessed) to the dpm or mean dpm of the concurrent vehicle control group.The approach to estimation of the relative skin sensitization potential was based on the mathematical estimation of the concentration of chemical necessary to obtain a threshold positive response (SI = 3); this is termed as the EC3 value. A substance was classified as a skin sensitizer if it induced a threefold or greater increase in local lymph node proliferative activity at one or more test concentrations when compared with concurrent vehicle-treated controls (SI≥3).

The relative potency index of test chemical was calculated to be 21.8.

Based on the relative potency index, test chemical was considered to be weak sensitizer.

This result is supported by another Local Lymph Node Assay (LLNA) conducted according to the method described in OECD Guideline 429. Groups of female CBA/CA mice (n= 5) were dosed topically on the dorsum of each ear with 25 ml of test material in 1:3 ethanol :diethyl phthalate. Each group received one of five test concentrations prepared as a w/v%. A concurrent vehicle control group was similarly treated with 1:3 ethanol: diethyl phthalate. Dosing occurred daily for three consecutive days. The animals received no treatment for two days and on the sixth day after the first application; all mice were injected intravenously via the tail vein with 250 ml of phosphate buffered saline containing 20 µCi of radiolabelled methyl thymidine (3HTdR). Five hours later, the mice were euthanized and the draining auricular lymph nodes were excised and pooled. Suspensions of the lymph node cells were prepared by mechanical disaggregation through 200-mesh stainless steel gauze. The cell suspensions were washed three times with phosphate buffered saline and precipitated overnight at 4 °C with 5% w/v trichloroacetic acid (TCA). The samples were then pelleted by centrifugation. The cells were re-suspended in 1ml of TCA and transferred to scintillation vials containing 10 ml of scintillation fluid. The incorporation of 3HTdR was measured by β- scintillation counting and expressed as disintegrations per minute (dpm) per lymph node.

The EC3 value obtained from LLNA assay performed to assess the sensitizing potential of test chemical was 5450 microgram/sq cm. Based on this value, the test chemical can be considered to be sensitizing to skin.

The results of the LLNA studies are supported by a Human Repeat Insult Patch Test [HRIPT] performed to evaluate the skin sensitization potential of the test chemical on 100 human volunteers. Each HRIPT study received approval from an independent ethical review committee (IRB institutional review board) prior to its initiation. During the induction phase an occlusive webril/adhesive patch (25mm Hill Top Chamber System) was used. 0.3 ml of the test material in 1:3 ethanol:diethyl phthalate (vehicle) was applied to each patch. The test material was allowed to volatilize for at least 15 min but no longer than 40 min prior to application to the skin. The left side of the back was used for the test area during the induction phase. Patches remained in place and were kept dry for approximately 24 h, after which time they were removed. A 24 h rest period, during which no test materials were applied. For alternate days test sites were observed and scored. The identical test site was then retreated until nine induction applications were completed over a period of approximately 3 weeks A rest period of approximately 2 weeks followed the last induction patch. No test materials were applied during the rest period. At the challenge phase, the original induction test sites were observed and each subject queried as to whether any reaction had been experienced during the rest period. The untreated right side of the back was used for the for the challenge phase. Patches were applied as in the induction phase and held in place for 24 h after which time they were removed and the challenge site scored. The original test sites were also observed. Scoring of the test sites was also carried out at 48, 72 and 96 hours.

The induction NOEL[No Observed Effect Level] derived from the Human Repeat Insult Patch Test [HRIPT] performed to determine the sensitizing potential of test chemical was70866 microgram/sq cm

Based on this value, test chemical can be considered to be sensitizing to skin.

These studies are supported by a Patch test performed to determine the degree of sensitization potential of the test chemical.

179 patients suspected of cosmetic allergy were patch tested with a series of 16 fragrances an-d 9 preservatives.

For patch testing, Silver patch testes (van der Bend bv, The Netherlands) were used. Reactions were read after 48 and 72 hours, and scored according to Internationally accepted criteria.

The test chemical was tested 10% in petrolatum.

2 of 179 patients tested gave a positive reaction to the test chemical.

Hence, the test chemical can be considered to be sensitizing to skin.

These results are further supported by a multi-centre patch test performed to determine the ingredients responsible for allergy to cosmetics in patients suffering from cosmetic related contact dermatitis. Most reactions (56.3%) were caused by skin care products, followed by nail cosmetics (13.4%), perfumes (8.4%), and hair cosmetics (5.9%). Preservatives were most frequently implicated (32.0%), followed by fragrances (26.5%) and emulsifiers (14.3%).

The multicenter study into the causative allergens in cosmetic products was initiated March 1, 1986, and ended July 31, 1987. Patch test procedures were carried out according to internationally accepted methods. Van der Bend patch test chambers (van der Bend, the Netherlands) were used for applying the allergens, and acrylate tape (Fixomull, Beiersdorf, Hamburg) for fixation.

 5% test chemical in petrolatum was applied to the skin of 147 volunteers. The patches were removed after two days and the reactions were read after 20 minutes and again one or two days later.

1% positive reaction was noted when tested in 147 volunteers.

Hence, the test chemical can be considered to be sensitizing to skin.

In another patch test 422 patients suspected of contact allergy to fragrances participated in the study.

Finn Chambers on Scanpor tape was used for patch testing, and the results were evaluated according to the recommendation of the International Contact Dermatitis Research Group.

5% concentration of the test chemical in petrolatum was used in the study to be most commonly applied to face and also to other body parts including neck, trunk, eyelid, lips, hand, arm, leg, scalp, ear, foot and widespread areas were also evaluated. Petrolatum was used as a vehicle control.

9 patients of the 422 tested positive for sensitization in the patch test. The test chemical showed a high number of positive responses (2.1%).

Hence, the test chemical was considered to be sensitizing to skin.

These results are further supported by a multicenter study extending over a three year period, dermatitis patients were tested for their sensitivity to fragrance materials. Test chemical was tested in 205 consecutive patients.

The test chemical was of high grade quality and provided by Haarmann and Reimer (Germany).It was dissolved in white petrolatum under gentle heating. Patch tests were performed with 1% and 5% test chemicalin white petrolatum using Finn Chambers® on Scanpor® applied for two days on the back. Reactions were recorded according to the ICDRG on days 2 and 3 or on days 2 and 4. Alpha-iso- methyl-ionone at both 1% and 5% concentrations produced a questionable/irritant reaction in 1 patient.

Hence, it can be considered to be a weak sensitizer to human skin.

The above human results are supported by a Mouse local lymph node assay (LLNA) test performed to investigate the skin sensitisation effect of the test chemical. The study was conducted as per OECD 429 Guidelines without any deviations. 4 mice per dose group were used for the study.

A daily topical application of 25µl of 10, 25, 50, 75, 100% test chemicalin EtOH:DEP (3:1) was made to the dorsal surface of each ear for 3 consecutive days. Control animals were treated with the vehicle alone. For each concentration of test chemical, a stimulation index (SI) relative to the concurrent vehicle-treated control was calculated.At least a 3-fold increase in lymph node cell proliferative activity was induced, compared to vehicle-treated controls (stimulation index SI ≥3). For positive LLNAs,an EC3 value was calculated which gave the estimated concentration of a chemical necessary to give a 3-fold increase in proliferative activity compared to vehicle- treated controls.

The EC3 value for the test chemical was 21.8 (5450µg/sq.cm). Based on the EC3 value the test chemical can be considered to be sensitizing to skin.

The above LLNA results are supported by a similar study performed as per OECD 429 Guidelines without any deviations. 4 CBA/J female mice per dose group were used for the study.

A daily topical application of 25µl of 10, 25, 50, 75, 100% test chemical in EtOH:DEP (3:1) was made to the dorsal surface of each ear for 3 consecutive days. Control animals were treated with the vehicle alone. For each concentration of test chemical, a stimulation index (SI) relative to the concurrent vehicle-treated control was calculated. At least a 3-fold increase in lymph node cell proliferative activity was induced, compared to vehicle-treated controls (stimulation index SI ≥3). For positive LLNAs,an EC3 value was calculated which gave the estimated concentration of a chemical necessary to give a 3-fold increase in proliferative activity compared to vehicle- treated controls.

The EC3 value for the test chemical was 21.8% (5450µg/sq.cm). Based on the EC3 value the test chemical can be considered to be sensitizing to skin.

The LLNA results are also supported by a Human Repeat Insult Patch Test conducted on 106 volunteers (26 males and 80 females) to determine the allergic potential of the test chemical. During the induction phase a 0.3 ml aliquot of 60% test chemical in 3:1 diethyl phthalate : ethanol was applied for 24 h to the back of each volunteer using a webril/adhesive patch (25 mm Hilltop Chambers®) under occlusion. A series of 9 induction patches were completed over a period of approximately 3 weeks. After a two week rest period a 24 h occluded challenge patch was applied to a virgin site. Reactions were evaluated at patch removal and then at 48, 72 and 96 h.

No sensitization reactions were observed in 106 volunteers tested. Hence, the test chemical was considered to be not sensitizing to human skin.

Even though some human studies claim that the test chemical was not sensitizing to skin, but majority of the results in mice and humans indicate that the test chemical has the potential to cause weak to moderate sensitization to skin. Hence, the test chemical can be considered to be sensitizing to skin.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Even though some human studies claim that the test chemical was not sensitizing to skin, but majority of the results in mice and humans indicate that the test chemical has the potential to cause weak to moderate sensitization to skin. Hence, the test chemical can be considered to be sensitizing to skin.