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Diss Factsheets

Administrative data

Description of key information

There was no evidence of sensitisation in the reliable studies available. However, the substance has a harmonised classification and as such is officially classified as a skin sensitizer under the Regulation (EC) No. 1272/2008 (Skin sens. 1B, H317).

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:
weight of evidence
Study period:
From 2000-03-14 to 2000-04-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 406 and in compliance with GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
17 July 1992
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
At the time of study performance, the LLNA method was not adopted.
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Davidson's Mill Farms, South Brunswick, NJ
- Age at study initiation: young adults
- Weight at study initiation: M: 390-505 g; F: 381-486 g
- Housing: group housed in suspended stainless steel caging with mesh floors
- Diet (e.g. ad libitum): Pelleted Purina Guinea Pig Chow #5025 ad libitum
- Water (e.g. ad libitum): Filtered tap water ad libitum
- Acclimation period: 18 days
- Indication of any skin lesions: No skin lesion identified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22 °C
- Humidity (%): 36-70 %
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2000-25-02 To: 2000-04-07
Route:
intradermal
Vehicle:
other: mineral oil
Concentration / amount:
40% w/v
Day(s)/duration:
Day 0
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, semiocclusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 %
Day(s)/duration:
Day 7 / 48 hours
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 %
Day(s)/duration:
Day 21 / 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
17 preliminary test animals, 20 test animals, 10 control animals
Details on study design:
RANGE FINDING TESTS:
- Intradermal injections: 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% and 0.1% w/w in 70/30 DEP/ETOH. 6 test sites/guinea-pig, 4 guinea-pigs. Due to severe irritation noted for all sites, 2 additional animals were injected with just the vehicle and exhibited the same severe response. One animal was used to evaluate the possible alternatives with propylene and mineral oil. Mineral oil was chosen for the intradermal injections to yield concentrations of 50%, 40%, 30%, 20%, 10% and 5%. The concentration which produced mild to moderate irritation was 40%.
- Topical induction: 100%, 75%, 50%, 25% w/w in 70/30 DEP/ETOH. 4 test sites/guinea-pig, 4 guinea-pigs. The concentration which produced mild to moderate irritation was 100%.
- Topical challenge: 100%, 75%, 50%, 25% w/w in 70/30 DEP/ETOH. 4 test sites/guinea-pig, 4 guinea-pigs. The highest concentration that produced responses in 4 guinea-pigs no more severe than 2 scores of 0.5 and 2 scores of 0 was 100%.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2, intradermal injections and topical application
- Exposure period: 48h for topical application
- Test groups:
INTRADERMAL: 3 pairs of intradermal injection (0.1 mL) in the shaved anterior shoulder on Day 0 after shaving as follows:
- 1/ 50% FCA
- 2/ test substance 40% in mineral oil
- 3/ test substance 40% in 50% FCA
SODIUM LAUREL SULFATE: As the substance is not a skin irritant, 24 hours prior to the topical application, the shoulder area was pre-treated with 5 %SLS mixture in petrolatum. Prior to the topical induction, the test sites were gently wiped with ethanol then water.
TOPICAL: 7 days after intradermal injections, 0.8 mL of the test substance (100%) was applied and covered with a gauze patch secured in place and wrapped with non-allergenic Durapore adhesive tape (semi-occlusive tape). After the 48 hour exposure period, the patches were removed and the test sites were wiped gently with DEP/ETOH then water, using a clean towel to remove any residual substance.
- Control group: similarly treated with the exception that the test substance was omitted.
- Evaluation (hr after challenge): approximately 24 and 48 hours after patch removal.

B. CHALLENGE EXPOSURE
- No. of exposures:
- Day(s) of challenge: 21 days after test initiation
- Exposure period: 24 hours
- Test groups: 0.4 mL of the test substance was applied using an occlusive 25 mm Hilltop Chamber®. The chambers were secured in place and wrapped with non-allergenic Durapore adhesive tape. After the 24 hours exposure period, the chambers were removed and the sites were gently wiped with DEP/ETOH then water, using a clean towel to remove any residual substance.
- Control group: similarly treated with the exception that the test substance was omitted
- Site: right side
- Concentrations: 100%
- Evaluation (hr after challenge): approximately 24 and 48 hours after patch removal.
Challenge controls:
None
Positive control substance(s):
yes
Remarks:
HCA (at least 85-91.7% pure), as an historical positive control
Positive control results:
INDUCTION PHASE: Severe erythema (3) was noted at all positive control sites during the topical induction phase.
CHALLENGE PHASE: All ten positive control animals exhibited signs of a sensitisation response (faint to severe erythema[1-3]) 24 hours after challenge. Similar indications persisted in 4 animals through 48 hours
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Very faint erythema (0.5) was noted in 8/20 animals
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
Irritation (grade 0.5) persisted in 5/20 animals
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25 %
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
faint to severe erythema[1-3]
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
25 %
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
very faint erythema (0.5) was noted in 5/10 animals
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Irritation (grade 0.5) persisted in 4/10 animals
Remarks on result:
no indication of skin sensitisation

Topical induction phases:

- Test animals (100% test substance): very faint to faint erythema (0.5 -1) in 9/20 animals 24 hours after removal of patch. Very faint erythema (0.5) persisted in 6/20 through 48 hours.

- Negative controls: very faint to faint erythema (0.5 -1) in 6/10 animals 24 hours after removal of patch. Very faint erythema (0.5) persisted in 4/10 through 48 hours.

- Positive control (100% HCA): Severe erythema (3) was noted at all positive control sites

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, ST 19 C 99 is not classified according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a dermal sensitisation study performed according to the EU test method B.6 and in compliance with GLP, ST 19 C 99 was tested in female Hartley guinea-pigs using the Guinea-Pig Maximisation Test method (20 treated animals + 10 controls).

The preliminary study determined the concentration which produced mild to moderate irritation to be used for the intradermal induction (40%) and for the topical induction (100%). 100% was selected as the highest non-irritant test substance concentration for the challenge.

 

ST 19 C 99 diluted in mineral oil at 40% (w/w) was administered by injection for intradermal induction. As the substance was not a skin irritant, 24 hours prior to the topical application, the site was pre-treated with 5% w/w sodium laurel sulphate in petrolatum. Topical induction was performed with ST 19 C 99 as supplied, 7 days after intradermal injections. For the challenge, ST 19 C 99 was tested at 100%.

 

The sensitivity of the guinea-pig is checked periodically at Product Safety Labs with HCA, a known sensitizer.

 

ST 19 C 99 did not produce evidence of skin sensitization during the study.

Under the test conditions, ST 19 C 99 is not classified according to the Regulation EC No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 1999-07-19 to 1999-08-20
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study performed according to OECD test guideline No. 406 and in compliance with GLP with acceptable restrictions: only few details on test animals and environmental conditions are given in the study report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
17 July 1992
Deviations:
yes
Remarks:
only few details on test animals and environmental conditions
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
At the time of study performance, the LLNA method was not adopted.
Species:
guinea pig
Strain:
other: Hartley derived
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Ace Animals Inc. Boyertown, PA 19512. USDA # 23-B-009
- Age at study initiation: no data
- Weight at study initiation: 300-500 g
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: at least 7 days
- Indication of any skin lesions: none indicated

ENVIRONMENTAL CONDITIONS
no data

IN-LIFE DATES: no data
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 %
Day(s)/duration:
Days 0, 7, 14 / 6 hours
Adequacy of induction:
other: Undiluted substance used
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: mineral oil
Concentration / amount:
50 %
Day(s)/duration:
Day 28
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
4 preliminary test animals. 20 test animals. 10 control animals
Details on study design:
RANGE FINDING TESTS: 100%, 75%, 50% and 25% in mineral oil. 4 guinea-pigs. Occlusive application, patches left in place for 6 hours. Reading at 24, 48 and 72 hours post application.
The highest concentration to cause mild irritation was determined to be 100%.
The highest non-irritating dose was 50%.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 hours
- Test groups: 0.5 mL of test material was applied to a one inch square gauze patch upon the clipped area of the flank, secured with Micropore tape, overwrapped with plastic and secured with Elastikon tape.
- Control group: similarly treated without the test item
- Site: clipped back
- Frequency of applications: once per week
- Duration: 3 weeks
- Concentrations: 100%
- Evaluation (hr after induction): 24 and 48 hours

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: after the 2 weeks rest period
- Exposure period: 6 hours
- Test groups: similarly than induction
- Control group: similarly treated without the test item
- Site: clipped back
- Concentrations: 50 % in mineral oil
- Evaluation (hr after challenge): 24, 48 and 72 hours

OTHER:
Challenge controls:
One group of 10 guinea-pigs not previously exposed to the test material and one group of 10 guinea-pig not previously exposed to DNCB.
Positive control substance(s):
yes
Remarks:
assessed every 6 months with DNCB
Positive control results:
Historical positive control data: DNCB at 0.3% concentration would be considered a sensitiser in albino guinea-pigs because there was 100% positive response at the 24 hour observation, during the challenge and 9/10 had a positive response at 48 hours.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
score of 0.5 in 15 animals
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
score of 0.5 in 4 animals
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
score of 0.5 in 5 animals
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.3%
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.3%
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

no other information

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, ST 19 C 99 is not classified according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a dermal sensitization study performed according to the OECD Guideline No. 406, ST 19 C 99 was tested in male and female Hartley derived guinea-pigs using the Modified Buehler method (10 treated animals + 10 controls).

 

Undiluted ST 19 C 99 was tested by occlusive epicutaneous induction. The guinea pigs were applied with the undiluted test substance once weekly during 3 weeks. A two-week rest period was followed by a challenge test consisting of applications of 50% test substance in mineral oil.

 

In all animals of both test and control groups, no significant reactions were observed at challenges sites of the skin at 24 and 48 hours after patch-removal. The positive control induced the appropriate response.

 

Under the test conditions, ST 19 C 99 is not classified according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
February 16, 2016
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Cf. Overall Remarks
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
27 June 2018
Deviations:
yes
Remarks:
Cf. overall remarks
GLP compliance:
no
Remarks:
Screening test
Type of study:
activation of keratinocytes
Details on the study design:
Test chemical
The 100X stock dilution was dissolved in DMSO to a final concentration of 200 mM. The test article was soluble in DMSO and formed clear solutions. From the stock 1the final 1X tested concentrations were: 2000, 1000, 500, 250, 125, 62,5 31,3 15,6 7.81, 3.91, 1.95 and 0.978 µM.

Test method conditions
- Description of test method used: KeratinoSensTM test method
- Cell line used, its storage conditions and source (e.g. the facility from which they were obtained): KeratinoSens cells were obtained from Givaudan. Upon receipt, the vial of cells was stored in a liquid nitrogen freezer
- Passage number and level of confluence of cells used for testing: passage number not reported. 70-90% confluence
- Cell counting method used for seeding prior to testing and measures taken to ensure homogeneous cell number distribution: a cell suspension of 10 000 cells / mL in assay medium was prepare using a Coulter counter. The stock of cell suspension was mixed often to ensure a uniform distribution of cells into each well.
- Luminometer used (e.g. model): Berthold Detection Systems luminometer initiated from an IBM-PC hosting the Windows-based Simplicity™ software. The light intensity in each well was measured at 565 nm in the form of relative light units (RLUs)
- Luciferase substrate used: ONE-Glo™ Reagent
- Demonstration of appropriate luminescence measurements: not included in the report
- The procedure used to demonstrate proficiency of the laboratory in performing the test method or to demonstrate reproducible performance of the test method over time: not included in the report

Test procedure
- Number of repetitions and replicates used; 1 repetition (negative), 3 replicates
- Test chemical concentrations and exposure time: from 0.98 to 2000 µM, 48 ± 1 hour
Description of evaluation and decision criteria used:
- Description of study acceptance criteria used;
1/ the EC1.5 value of the positive control should be within two standard deviations of the historical mean of the testing facility
2/ The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20%
3/ the luciferase activity induction obtained with the positive control, cinnamic aldehyde, should be statistically significant above the threshold of 1.5 below 64 µM in each definitive assay
- Description of any modifications of the test procedure:
On Luciferase activity measurements: Promega ONE-Glo was added for 5 minutes incubation in the dark, and then reading was done within 45minutes.
In the OECD TG: “After the 48 hour exposure time with the test chemical and control substances in the KeratinoSensTM test method, cells are washed with a phosphate buffered saline, and the relevant lysis buffer for luminescence readings added to each well for 20 min at room temperature. Plates with the cell lysate are then placed in the luminometer for reading which in the KeratinoSensTM test method is programmed to: (i) add the luciferase substrate to each well (i.e. 50 µl), (ii) wait for 1 second, and (iii) integrate the luciferase activity for 2 seconds. In case alternative settings are used, e.g. depending on the model of luminometer used, these should be justified. Furthermore, a glow substrate may also be used provided that the quality control experiment of Annex 3 is successfully fulfilled”
Positive control results:
EC 1.5 = 10.37. Mean IC 50 > 64 µM
Key result
Run / experiment:
other: Run 1 / mean of 3 experiments
Parameter:
other: Imax
Value:
1.07
Vehicle controls validity:
not specified
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Run 1 / mean of 3 experiments
Parameter:
other: EC1.5
Value:
2 000
Vehicle controls validity:
not specified
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Run 1 / mean of 3 experiments
Parameter:
other: IC50
Value:
23.22
Vehicle controls validity:
not specified
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Run 1 / mean of 3 experiments
Parameter:
other: IC30
Value:
20.517
Vehicle controls validity:
not specified
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: Not included in the report

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not reported
- Acceptance criteria met for positive control: The luciferase activity induction obtained with the positive control, Cinnamic Aldehyde, was above the threshold of 1.5-fold.
- Range of historical values if different from the ones specified in the test guideline:
Not reported but the EC1.5 value was within two standard deviations of the historical mean of the OECD TG (e.g. between 7 μM and 30 μM based on the validation dataset).


Interpretation of results:
study cannot be used for classification
Executive summary:

The test item was evaluated for the ability to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in a KeratinoSens(TM) test similar to the OECD Guideline No. 442D. In this test, KeratinoSens(TM) cells cultures were treated with 12 concentrations of test chemical (0.978, 1.95, 3.91, 7.81, 15.6, 62.5, 125, 250, 500, 1000 and 2000 µM) and control substances (positive control: Cinnamic Aldehyde in DMSO, vehicle control: 1% DMSO in exposure medium, blank) and were then incubated for about 48 hours at 37 +/-1.0°C in presence of 5% CO2. Luciferase activity (Imax and EC1.5) was assessed using a luminometer. One experiment was performed.

The test item has a mean IC50 of 23.22 µM . The maximum luciferase activity induction (Imax) was 1.07-fold. The IC1.5 was reported to be above 2000 µM.

Overall, the test item is classified as negative in the KeratinoSensTM assay.

The acceptance criteria were met for the positive control.

However, the reported effective concentration showing 50% increase of luciferase induction relative to solvent control (EC1.5 > 2000 µM) is doubtful because the viability was 8.85% at 31.3 µM and then 0.0 for higher concentrations. In addition, according to the OECD TG, a negative result obtained with concentrations < 1000 μM should also be considered as inconclusive. Therefore, this study cannot be used for classification.

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

Various studies were available regarding the skin sensitisation potential. They are listed in the Table below.

 

Table 7.4.1 / Sumary of Skin sensitisation tests

 

Ref.

Test Method

Test Conclusion

Klimisch score

1

PSL, 2000

GPMT (OECD 406 / GLP)

Negative

1

2

Celsis 1999

Buehler (OECD 406 / GLP)

Negative

2

3

Toxicol, 1991

GPMT (OECD 406 / GLP)

Positive

3

4

BRT 2000

LLNA (OECD 429 / GLP)

Positive

3

5

BRT 2003

Ear Swelling Test (non GLP)

Positive

3

6

IIVS 2016

KeratinoSens (eq. OECD 442D, non-GLP)

Negative

4

7

BASF 2017

hCLAT (eq. OECD 442E / GLP)

Positive

3

8

TKL 1995

HRIPT

Negative

2

9

TKL 1999

HRIPT

Negative

2

 

1/ The study of PSL (2000) was conducted according to the OECD test guideline No. 406 and in compliance with GLP without any deviation. In this study, the substance was tested in guinea-pigs using the Guinea-Pig Maximisation Test method (20 treated animals + 10 controls).This study also used Hilltop chambers to apply the non-injected exposures (0.4 mL).The preliminary study determined the concentration which produced mild to moderate irritation to be used for the intradermal induction (40%) and for the topical induction (100%). 100% was selected as the highest non-irritant test substance concentration for the challenge. The substance did not produce evidence of skin sensitization during the study.

 

2/ The study of Celsis (1999) supports the conclusion of PSL (2000). The study was conducted according to the OECD test guideline No. 406 and in compliance with GLP, using the Modified Buehler Method (10 treated animals + 10 controls). 100% was selected as the highest dose for induction, and 50% in ethanol as the highest non-irritant test substance concentration for the challenge. The substance did not show evidence of sensitisation during the study.

 

3/ The study of Toxicol (1992) was disregarded. Indeed, even if it was conducted according to the EU test method B.6 and in compliance with GLP, this study is not considered as acceptable as several major deviations were noted:

- sodium lauryl sulphate was not used to create a local irritation, while the substance was reported as not a skin irritant in this study.

- the first challenge indicated that 12 guinea pigs had been sensitised. However, the nature of the reactions (fading at the later time point in 5 cases) suggests they may in fact be due to skin irritation. Rechallenge under identical conditions on the opposite flank shows that the responses were not reproducible in the guinea pigs, only one guinea-pig showing a reaction. Thus, despite any evidence of irritation in controls under the conditions of this study, the reactions in the test animals does not seem to be of an allergic nature. This lack of reproducibility may be due to the detachment of patches observed during the first challenge.

- it neither includes positive controls nor historical positive control data. Therefore, the sensitivity and reliability of the experimental technique used cannot be assessed.

Based on these deviations this study was disregarded.

 

4/ The study of BRT (2000) was disregarded.The study used the test material at three concentrations, 25, 50 and 100% using acetone/olive oil (4:1) as the vehicle. The 25µL doses were applied daily for three days, and all three concentrations induced evidence of irritation (redness and ear swelling). The stimulation index (SI) values were 9.0, 21.4 and 23.7 for the 25, 50 and 100% concentrations respectively and the study concluded that the substance is a skin sensitizer under the test conditions.Even if it was conducted according to the OECD test guideline No. 429 and in compliance with GLP, this study is not considered as acceptable. Indeed, the ear swelling and erythema observations were not quantified, therefore it cannot be known whether the level of irritation induced by the substance exceeded the threshold of 25% increase in ear thickness and/or erythema score ≥3, that have been identified as being likely to be result in a false positive due to irritation.

 

5/ The study of BRT (2003) was disregarded. This study is an adaptation of the LLNA assay designed to discriminate between false positive irritant substances and true skin sensitizers (Gerberick, 2002. The mice are exposed to the test substance in the same way as in the LLNA but the ears are measured for changes in ear thickness (swelling) and the lymph node cells are counted and measured for the relative frequency of B220+ cells using flow cytometry; a ratio of greater than 1.25 B220+ cells is considered to be a sensitizer and less than 1.25 an irritant. Sodium lauryl sulphate was used as an irritant false positive control and isoeugenol used as a true positive control. Unfortunately, the results of the study were inconclusive for several reasons. The standard vehicle control (acetone:olive oil) induced an 11.8% increase in ear thickness, which is not normally observed. SLS produced both an increase in cell numbers and provided a ratio result of >1.25 that indicated sensitizing potential. The substance induced an increase in ear thickness, but not in a dose related pattern, and an increase in the ratio greater than 1.25, but in the absence of an increase in cell numbers. Because of these technical problems it is considered that this study is not reliable. Furthermore, the publication of Gerberick et al., (2002) showed that one of five irritants tested (salicylic acid) induced a ratio >1.25 in 2/4 experiments. They also showed that cell numbers were increased by both irritants and sensitizers, although generally to a greater extent by sensitizers. Thus, it seems clear that there must be an increase in lymph node cell proliferation in order for any change in the ratio of B220+ cells to have a toxicological significance; no such proliferation was observed with exposure to the substance in this study.

 

6/ The study of IIVS (2016) supports the conclusion of PSL (2000). The KeratinoSens study was conducted similarly to the OECD test guideline No. 442D and in compliance with GLP. The test showed that the substance was very toxic to the cells and the viability was estimated to be 94% at 3.7 µg/mL and 3% at 7.4 µg/mL. Exposure to the substance caused no induction of the ARE gene and the substance was predicted not to be a sensitizer. The substance is not within the applicability domain of the method, therefore negative result obtained with concentrations < 1000 μM should also be considered as inconclusive. In addition, some information on the test methods and validation criteria were missing. This study can only be used as supporting study.

 

7/ The study of BASF (2017) was disregarded. Even if this hCLAT study was conducted similarly to the OECD test guideline No. 44E and in compliance with GLP, supporting evidence that the dose levels selected for use in this study were excessively toxic can be taken from other in vitro studies performed on this substance:

- The dose levels used in this study were much higher than those that were shown to be highly toxic in the Keratinosens assay. The IC50 (50% viability) in the Keratinosens assay was 5.5 µg/ml, whereas in the h-CLAT the CV75 was 28 -33 µg/ml, but the CV50 was >500 µg/ml

- In a mouse lymphoma assay a dose level of 25 µg/mL exceeded 90% toxicity.

Based on these comparisons, the toxicity dose-response curve observed in the hCLAT study is not considered to be plausible. This study is therefore considered to be unreliable.

 

8-9/ Two occlusive repeated insult patch tests, conducted in more than hundred volunteer subjects each, did not show any evidence of sensitisation when tested at 10% or 20% in diethyl phthalate (TKL, 1995 & 1999).

 

In conclusion, there was no evidence of sensitisation in the reliable studies available. However, the substance has a harmonised classification and as such is officially classified as a skin sensitizer under the Regulation (EC) No. 1272/2008 (Skin sens. 1B, H317).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonized classification:

The substance is classified as skin sensitiser:

- Skin Sens. 1B (H317: May cause an allergic skin reaction.) according to the Regulation (EC) No. 1272/2008 (CLP) including ATP3 and to the GHS.

No information is available regarding respiratory sensitisation.