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Administrative data

Description of key information

The test substance, GL500, did not produce skin sensitization under the conditions of the in-vivo (non LLNA study).

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
Study period:
19 July 2013 - 01 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK.
- Females nulliparous and non-pregnant: yes
- Age at study initiation:eight to twelve weeks old.
- Weight at study initiation:15 to 23 g
- Housing:The animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum):Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
- Acclimation period:Five days


ENVIRONMENTAL CONDITIONS
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25 °C and 30 to 70%, respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Undiluted test item was used as well as the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1.
No. of animals per dose:
4 animals per dose.
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility:The mouse was treated by daily application of 25 µL of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
- Irritation:Local skin irritation was scored daily according to the scale
- Systemic toxicity:Any clinical signs of toxicity, if present, were also recorded. The body weight was recorded on Day 1 (prior to dosing) and on Day 6.
- Ear thickness measurements:The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose on Day 1, post dose on Day 3 and on Day 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.
- Erythema scores:not specified

MAIN STUDY


ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method:Skin Sensitization: Local Lymph Node Assay
- Criteria used to consider a positive response:The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3 HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3 HTdR incorporation will be classified as a "non-sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION:

For the purpose of the study, the test item was used undiluted and freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced a suitable formulation at the required concentration.The test item was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.


Groups of four mice were treated with the undiluted test item or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.

A further group of four mice received the vehicle alone in the same manner.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Key result
Parameter:
SI
Value:
3.61
Remarks on result:
positive indication of skin sensitisation based on QSAR/QSPR prediction
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: not specified.

DETAILS ON STIMULATION INDEX CALCULATION: Please see table in results section.


EC3 CALCULATION: The concentration of test item expected to cause a 3 fold increase in 3 HTdR incorporation (EC3 value) was calculated to be 70%.


CLINICAL OBSERVATIONS:There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS: Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Concentration(%v/v) in acetone/olive oil 4:1

Stimulation Index

Result

25

2.88

Negative

50

2.61

Negative

100

3.61

Positive

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The test item was considered to be a sensitizer under the conditions of the test.
Executive summary:

Introduction

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

 

Methods

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of four animals was treated with acetone/olive oil 4:1alone.

Results

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Please see table in results section.

The concentration of test item expected to cause a 3 fold increase in3HTdR incorporation (EC3value) was calculated to be 70%.

 

 

Conclusion

The test item was considered to be a sensitizer under the conditions of the test.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 November 2013 - 24 January 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
17 July 1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The LLNA study was conducted however there was concern that the results from this study may not be accurate therefore tihis second study using alternative methodology (GPMT) was conducted for confirmation.
Additionally it should be noted that testing was conducted to satisfy regulatory purposes outside of REACH.
Specific details on test material used for the study:

Lot No. 20130510
Appearance Colorless liquid
Ingredient Dibutyl terephthalate, Butyloctyl terephthalate, Dioctyl terephthalate
Molecular weight 350
Purity 99.81%
pH 6.5
Affinity Hydrophobic, lipophilic
Stability Stable at room temperature
Date of manufacture May 10, 2013
Expiration date May 10, 2018
Storage condition Room temperature (18.2 – 21.0°C)
Handling instruction Not specified
Supplier
Name LG Chem, Ltd.
Address LG Twin Towers, 20, Yeouido-dong, Yeongdeungpo-gu, Seoul, 150-721, Korea
Date of receipt Nov. 4, 2013
Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Central Lab. Animal Inc., Korea
- Age at study initiation: 4-5 weeks old.
- Weight at study initiation: 249 - 396 g
- Housing:The animals were individually housed in Stainless wire mesh cages, 210W×350D×180H (mm).
- Diet (e.g. ad libitum): Free access to mains tap water and food (Purina experimental diet for guinea pig 38065 supplied by Cargill Agri Purina Inc) was allowed throughout the study.
- Acclimation period: Four days


ENVIRONMENTAL CONDITIONS
The temperature and relative humidity were controlled to remain within target ranges of 19 to 22 °C and 49 to 70%, respectively. The rate of air exchange was 10-15 changes per hour and the lighting was controlled by automatrd timer to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness, intensity 150 - 300 Lux.
Route:
intradermal
Vehicle:
paraffin oil
Concentration / amount:
100, 50, 25, 12.5, 6.25, 3.13 w/v%
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% test substance
Day(s)/duration:
2
Adequacy of induction:
highest technically applicable concentration used
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% test substance
Day(s)/duration:
1
Adequacy of challenge:
not specified
No. of animals per dose:
20 for test substance
10 for control substance
Details on study design:
Preliminary Study
The preliminary portion of the study was conducted under Non GLP condition in order to select appropriate concentrations for induction and challenge.

Method
The shoulder region of two guinea pigs was clipped (SM-110, JOAS, Korea) and shaved (SR-292S, JOAS, Korea) free of hair. Six sites, three each on the left and right sides of the midline of the shaved shoulder region were selected as injection sites. Each animal was injected intradermally with 0.1 mL of the test substance at dose levels of 100, 50, 25, 12.5, 6.25, and 3.13% using 1-mL disposable syringes (26G needle). The highest concentration was administered in the top left area followed by counter clockwise injections in subsequent injection sites.
The left and right flanks of two additional animals were carefully clipped and closely shaved free of hair. Six areas (2×2 cm), three left and three right sides of the midline, were selected as application sites. For the topical applications, 0.1 mL of each test substance concentration (100, 50, 25, 12.5, 6.25, and 3.13%) was placed on a 2×2 cm patch (PIP-Tokyo Co., Ltd., Japan), covered with 5×5 cm surgical tape (3MTM
Blenderm surgical tape, 3M Japan) and placed on the application sites; higher concentrations to the left and lower concentrations to the right. Animals were wrapped with 7.5 cm adhesive tape (Silkytex, Alcare Co., Ltd., Japan) and secured with surgical tape (Steri-Drape No. 2050, 3M Co., Ltd., Korea) for a 24-hour occlusive dressing. Then, the occlusive dressings were removed and any residual test substance was removed from the application sites by wiping gently with absorbent cotton (DaeHan Medical Supply Co., Ltd., Korea) moistened with tepid water. Two animals, to which each concentration of the test substance was applied to each animal, were used.

Observation of skin reactions
The skin reactions were evaluated at 24 and 48 hours after injection and at 24 and 48 hours after patch removal according to the evaluation standard of skin reactions by Magnusson and Kligman1).

Justification for selection of induction and challenge dose levels
As a result of the preliminary study, at 24 and 48 hours after intradermal injection, there were no adverse skin reactions at 100, 50, 25, 12.5, 6.25 and 3.13% test substances in both animals.
The topical application sites of both animals at 100, 50, 25, 12.5, 6.25 and 3.13% test substances did not reveal any evidence of adverse skin reactions at 24 and 48 hours after patch removal .
The dose level for the first induction was the highest concentration of the test substance which did not produce necrosis at the intradermal injection sites. Therefore, the dose level was selected at 100% test substance. Also, the dose level for the second induction was the highest concentration of the test substance which produced mild to moderate skin reactions at the topical application sites. However, no skin reactions were evident at the topical application sites at any concentration of the test substance. Therefore, the dose level was selected at 100% test substance. The challenge dose level was selected at 100% test substance, at which no adverse skin reactions were produced at the topical application sites.

Dosing
Route
First induction: Intradermal route
Second induction and challenge: Dermal route

Justification for selection
The intradermal and dermal routes were chosen to evaluate sensitivity of the test substance.

Method
First induction
The day of the first induction was designated as Day 0. On the day prior to the first induction, the shoulder region of guinea pigs was clipped and shaved free of hair. A 2×4 cm area on the shaved shoulder region was utilized as the application site. Six injection sites (2×4 cm application sites), three on each side of the midline of the shaved dorsal surface were utilized for the intradermal injections. A volume of 0.1 mL of each dose was injected using 1-mL disposable syringes (26G needle).

10% SDS application
Since the application sites in the preliminary topical phase of the study did not reveal any indication of skin reactions, the application sites of all animals were trimmed with electric clippers and closely shaved with an electric shaver on Day
5. The application sites were pretreated with 0.5 mL of 10% SDS using 1-mL disposable syringes prior to the topical induction on Day 6.

Second induction
On Day 7 (24 hours after SDS topical application), any remaining SDS was wiped off at the application sites with absorbent cotton moistened with tepid water. A volume of 0.2 mL of the 100% test substance on a 2×4 cm patch (TegadermTM+Pad, No.3582, 3M Co., Ltd., U.S.A.) was applied to the shoulder
region of application sites of the test substance group. A volume of 0.2 mL of liquid paraffin was applied to the application sites of the control group using 1-mL disposable syringes. Patches were wrapped with 7.5 cm expandable adhesive tape and secured with surgical tape for a 48-hour occlusive dressing. Wrappings and patches were removed at 48 hours after application. Then, the application site was wiped gently clean with absorbent cotton moistened with tepid water.

Challenge
On Day 20, the left and right dorsal application sites were carefully trimmed with electric clippers and closely shaved with an electric shaver.
On Day 21, a volume of 0.1 mL of the 100% test substance on 2×2 cm patch was applied to the left challenge sites of the test substance and control groups. At the right challenge sites of the test substance and control groups, a volume of 0.1 mL of liquid paraffin was applied using 1-mL disposable syringes. Patches were wrapped loosely with 7.5 cm expandable adhesive tape and secured with surgical tape as a 24-hour occlusive dressing. Wrappings and patches were removed at 24 hours after application. Then the application sites were wiped clean by absorbent cotton moistened with tepid water. Rechallenge was not conducted.

Parameters Evaluated

Clinical signs
All animals were observed for general condition and clinical signs once daily throughout the study.

Body weights
Body weights were recorded on the day of initiation (Day 0), once weekly and on the day of final dermal observations (Day 24).

Evaluation of skin sensitization
Skin reactions such as redness and swelling were recorded at 24 and 48 hours after challenge patch removal according to the evaluation standard of skin reaction (Method of Magnusson & Kligman)1). The application sites were shaved with an electric shaver at approximately 3 hours prior to observations. Representative photographs were taken from each group at 24 and 48 hours after challenge patch removal.

Evaluation Standard of Skin Reaction (Method of Magnusson & Kligman)
Evaluation standard of skin reaction Score
No reaction … 0
Scattered mild redness 1
Moderate & diffuse redness 2
Intense redness & swelling 3

Classification of sensitization
Individual scores were added at each interval and divided by the number of animals to determine the mean skin reaction score for each group. In addition, sensitization rate was calculated and graded according to the classification of Magnusson & Kligman to classify sensitization grade.
Sensitization Rate % = (Number of animals with positive reaction) / (Number of animals testd) x 100

Classification of sensitization grade of Magnusson & Kligman
Sensitization rate (%) Grade Class
0*–8 I Weak
9–28 II Mild
29–64 III Moderate
65–80 IV Strong
81–100 V Extreme
*Although the sensitization rate of 0% is classified as ‘weak’ in the reference, it is classified as ‘negative (no sensitization)’ when the sensitization rate is 0% in this study.


Challenge controls:
Liquid Paraffin
Positive control substance(s):
yes
Remarks:
The positive control study was conducted routinely using 1-chloro-2,4-dinitrobenzene (CDNB), a known positive control substance using Maximization test1). Consequently, the experimental method used for skin sensitization study was considered to be valid
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No abnormal signs or symptoms were evident in any animals throughout the course of the study
Remarks on result:
no indication of skin sensitisation

Body Weights

 

All animals exhibited normal body weight gain. Mean body weight gains were 168 g and 166 g in the test substance and control groups, respectively.

Observations of Skin Reactions

In the test substance group, the 100% test substance and liquid paraffin challenge sites did not reveal any evidence of adverse skin reactions such as redness and swelling at 24 and 48 hours after challenge patch removal in any animal (Mean score 0 each).

In the control group, the 100% test substance and liquid paraffin challenge sites did not reveal any evidence of adverse skin reactions such as redness and swelling at 24 and 48 hours after challenge patch removal in any animal (Mean score 0 each).

Sensitization Rate and Grading

The sensitization rates in the 100% test substance and liquid paraffin challenge sites of the test substance and control groups were 0% at 24 and 48 hours after challenge patch removal, respectively

Therefore, the sensitization grade was classified to be ‘I’, ‘negative (no sensitization)’, according to the classification of sensitization grade of Magnusson &Kligman1).

For the background data, 1-chloro-2,4-dinitrobenzene (CDNB) was applied to the application sites for the positive control animals periodically. The sensitization rate of CDNB was 100% at 24 and 48 hours after patch removal, and the sensitization grade was classified to be ‘V’, ‘Extreme’. Consequently, the experimental method used for the skin sensitization study was considered to be valid.

Interpretation of results:
GHS criteria not met
Conclusions:
The test substance, GL500, did not produce skin sensitization under the conditions of this study.
Executive summary:

The purpose of this study was to evaluate the skin sensitization potential of the test substance, GL500, in Hartley guinea pigs using Maximization Test1). Test groups consisted of the test substance group (20 animals) and control group (10 animals). Since topical application in the preliminary study did not reveal any evidence of adverse skin reactions, the application sites of all groups were pretreated with 10% sodium dodecyl sulfate prior to the second induction.

 

 

In the test substance group, the 100% test substance was injected intradermally for the first induction. The second induction was conducted with the 100% test substance occluded for 48 hours. The challenge was conducted with the 100% test substance and liquid paraffin occluded for 24 hours. No skin reactions such as redness and swelling were evident at the challenge sites of the 100% test substance and liquid paraffin at 24 and 48 hours after challenge patch removal in any animal.

In the control group, the first and second inductions with liquid paraffin and the challenge with 100% test substance and liquid paraffin were conducted. No skin reactions such as redness and swelling were evident at the challenge sites of the 100% test substance and liquid paraffin at 24 and 48 hours after challenge patch removal in any animal.

During the observation period, no abnormal clinical signs or body weight gain was evident in any animals of any groups.

 

 

Based on the results of this study, the test substance, GL500, did not produce skin sensitization under the conditions of this study.

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

Justification for classification or non-classification

Harlan Study number 41302153

From the results of a LLNA study the test material is regarded as a sensitiser and the EC value was calculated to be 70% w/w.

 

An in depth analysis of the results showed that the dose response was rather shallow (SI’s of 2.88, 2.61, and 3.61 were obtained at test concentrations of 15%, 50% and 100% respectively), with the intermediate concentration of 50% being slightly lower than at 25%. All responses were very close to the SI of 3 which is the criterion for classification as a skin sensitiser according to EU CLP and UN GHS. The EC3 was calculated to be 70% by extrapolation. According to EU CLP, a substance requires classification as Category 1A if the EC3 is ≤ 2%, or as Category 1B if the EC3 is > 2%. According to Basketter et al (2005), substances giving an EC3 value >2% in the LLNA can be regarded as having moderate skin sensitising potency and according to ECETOC Technical Report No. 87 (2003), substances with an EC3 > 10% can be regarded as weakly sensitising. The EC3 of 70% for GL500 therefore indicates that if the substance is a true skin sensitiser, it is only of low skin sensitising potency.

A slight increase in ear thickness (13.95%) was noted in the single mouse treated with undiluted test item in the preliminary screening test of the LLNA. An increase of ≥25% ear thickness is considered excessive. Whilst ear thickness was not measured in the main study of the LLNA, and no signs of irritation were detected in any animal by visual examination, the increase in ear thickness in the preliminary test indicates that some local inflammatory response had occurred. Together with the shallow and non-linear dose response in the LLNA, the possibility that the > 3 SI response was due to inflammatory effects (due to slight irritation), rather than skin sensitisation, cannot be ruled out.

Additionally from the study data the mean DPM value of 662.66/ lymph node for the vehicle (negative) control animals appears to be low. When this is compared to the historical control data it is indeed low compared to the mean DPM/node for animals treated with acetone:olive oil (4:1) during 2013, which is about 1200. If the mean DPM/lymph node for the vehicle control animals had been above 796, the result of the LLNA would have been negative.

 

This suggests that there is a good case for the result of the LLNA to be considered a false positive result particularly when compared to the negative (not sensitising) GPMT result below.

Biotoxtech Study number B13872

In a Guinea Pig Maximisation Test (GPMT) study GL500 did not produce skin sensitisation.

Considering the two studies the GPMT is considered the most reliable result and that there is a strong possibility that the LLNA result may be a false positive result.

Based on the information above it can be concluded that the test substance GL500 is not classified as a sensitiser.