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Diss Factsheets

Administrative data

Description of key information

There are two in vivo skin sensitisation studies available on the target substance, a LLNA and a GPMT. There is also old human volunteer data. In addition, there is an in vivo study (GPMT) on an analogue substance.

The substance was tested as a 40% aqueous solution in a LLNA. The substance gave a positive response (SI >3) at all dose concentrations (5, 10 and 25% as test material, equivalent to 2, 4 and 10% as substance). An additional Guinea Pig Maximisation Test was recently undertaken to confirm or refute the findings of the LLNA study. In the GPMT, no responses in the substance-induced animals exceeded those of the vehicle-induced group animals and hence the substance is not considered to be a dermal sensitiser. The GPMT is considered to provide the definitive result for classification purposes.

A structural analogue was also negative for skin sensitisation in a GPMT.

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study carried out according to OECD 429 (2010) and GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands
- Age at study initiation: 8-10 w
- Weight at study initiation:
- Housing: individually in Markolon Type I with wire mesh top
- Diet: ad libitum, pelleted standard diet
- Water: ad libitum, tap water
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12

Vehicle:
dimethylformamide
Concentration:
5%, 10% and 25%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: a solubility experiment was carried out prior to the main study
- Irritation: pre-test performed in 2 female animals with the concentrations of 10, 25, 50 and 100%, each applied on the ear for 3 consecutive days. At 25 and 50% slight redness was seen, as well as at 100% were it was more pronounced. Thus, 25% was chosen as the highest concentration for the main study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Lymph node proliferation response: No of radioactive disintegrations/ min/ lymph node (DPM)
- Stimulation index: 3HTdR incorporated into lymph node cells treated animals/control
- Criteria used to consider a positive response: 1. Exposure to at least one concentration resulted in at least a 3-fold increase of 3HTdR incorporation; 2. Data with a dose-response

TREATMENT PREPARATION AND ADMINISTRATION:
Each test group was treated by topical application to the dorsal surface of each ear lobe with the relevant concentrations in dimethylformamide, with an application volume of 25 ul, once daily for 3 consecutive days. A vehicle control group was also used. The preparations were made freshly before each dosing. Five days after the first epidermal application, all mice were intravenously injected with 250 ul of 81.1 uCi/ml 3HTdR. Five hours after this treatment, the animals were sacrifised, and the draining lymph node were used for the evaluation of cells and determination of the Stimulation Index. The level of 3HTdR incorporation was measured with a beta-scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean and standard deviations were calculated for the body weights.
Parameter:
SI
Remarks on result:
other: Vehicle control: - 5%: 3.64 10%: 7.45 25%: 11.08
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle control: 3338 5%: 11993 10%: 24517 25%: 36420
Key result
Parameter:
SI
Value:
ca. 3.64
Test group / Remarks:
2: 5%
Key result
Parameter:
SI
Value:
ca. 7.45
Test group / Remarks:
3: 10%
Key result
Parameter:
SI
Value:
ca. 11.08
Test group / Remarks:
4: 25%

Table 1: Calculation and results of individual data.

Concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-BG

No of lymph nodes

DPM/lymph node

S.I.

-

BG I

59

-

-

-

-

-

BG II

52

-

-

-

-

-

1

3338

3283

8

410.3

1

5

2

11993

11938

8

1492.2

3.64

10

3

24517

24462

8

3057.7

7.45

25

4

36420

36365

8

4545.6

11.08

BG: Background (1 ml 5% trichloroacetic acid) in duplicate

1: vehicle control group

2-4: test groups

S.I.: Stimulation Index

No deaths occured during the conduction of the experiment. The animals did not show any treatment related clinical signs, except for redness detected in the application site, after the third dosing of the highest concentration. The body weights were within the normal range (details in the attached document).

The results for the positive control confirm the validity of this test (details in the attached document).

Interpretation of results:
study cannot be used for classification
Conclusions:
The test material RL 860/07 was a skin sensitizer in this LLNA test. Since RL 860/07 contains 40% CAS 10595-49-0, it can be concluded that CAS 10595-49-0 is a skin sensitizer.
Executive summary:

In the present Local Lymph Node Assay (LLNA), the test material RL 860/07 (40% CAS 10595 -49 -0 in water) dissolved in dimethylformamide was assessed for it skin sensitizing potential. The concentrations 5, 10, and 25% (w/v) were applied on the ear of female CBA/CaOlaHsd mice, once per day, for three consecutive days (4 animals/group). Vehicle controls were tested only with dimethylformamide. The concentrations chosen were based on a range-finding study performed prior to the main test. Hexyl cinnamic aldehyde was used as a positive control. Five days after the first treatment, the animals were injected intravenously with radiolabelled thymidine and were sacrificed after five hours.The proliferative capacity of the lympocytes in draining lymph nodes was examined. The results revealed Stimulation Indices higher than 3, increasing on a dose-dependent manner. On the basis of this result, the test material RL 860/07, and hence, CAS 10595 -49 -0, shall be classified as skin sensitizers.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2nd August 2016 - 2nd September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
The gauge of needle used to dose the animals for the first intradermal screen phase was not recorded. There were no unusual local effects observed. The size of the needle for Induction A (26-gauge) was recorded.
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A LLNA assay has already been conducted which gave a positive response. However the registrant is concerned that the result was in fact a false positive. The reasons for the concerns are listed below:

1. The chemical structure does not contain any obvious structural features which are normally associated with skin sensitization ie the presence or metabolic/non-metabolic formation of protein reactive functional groups.

2. The structurally related substance N,N,N-trimethyl-3-(undec-10-enoylamino)propan-1-aminium methyl sulfate (CAS 94313-91-4, EC 304-990-8) has been tested in a reliable Guinea Pig Maximisation Test (GPMT) and gave a negative response.

3. There is significant discussion in the toxicology literature regarding the validity of positive results obtained in the LLNA on irritant, surfactant substances. One of the more recent references (Ref 1, Ball et al 2011) notes that:

Since its introduction, there have been publications reporting apparent false positive results from the LLNA for certain groups of chemicals, including surfactants (Garcia et al., 2010; Kreiling et al., 2008; Woolhiser et al., 1998), the conclusion being that the LLNA may in some cases be misrepresenting the sensitizing potential of the tested substances. The basis for claims that the assay gives ‘false positives’ comes from contradictory data from experimental results obtained from other animal models, e.g. the GPMT, human data (hRIPT, worker/consumer experience), and the lack of structural alerts within the substance that are associated with sensitizing potential.

The paper also recommends that future in vivo testing of surfactants for sensitizing potentials should be done using one of the available guinea pig test guidelines to insure the highest relevance of the results to humans.

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Screening study: 0.5 - 100%.
Main study: Induction A - 2.5 or 5%; Induction B - 100%.
Day(s)/duration:
Single application
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Main study: 5%.
Day(s)/duration:
24 hours occlusion following a single topical application.
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Screening study: 3 animals/sex/dose
Main study: 5 animals/sex/dose
Details on study design:
There are two stages to the maximization test. The first stage is the induction and consists of intradermal injections followed in seven days by a topical application of the test article. The second stage is the challenge which consists of a topical application performed 14 days following completion of the induction phase.
Challenge controls:
Vehicle control: Distilled water
Positive control substance(s):
yes
Remarks:
Historical data for 2-mercaptobenzothiazole 98%
Positive control results:
Mercaptobenzothiazole was confirmed to be GHS Category 1B dermal sensitiser using the Guinea Pig Maximization Test.
Key result
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
Erythma: Discrete or patchy erythema Dermal Observations: None
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
72
Group:
test chemical
Dose level:
100%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
Erythma: Discrete or patchy erythma. Dermal Observations: None
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
72
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
100%
No. with + reactions:
4
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
100%
No. with + reactions:
2
Total no. in group:
20

 Group  Mixture  Time Point  Erythma  Dermal Observations
 Induced Group  TA  48 hrs  Discrete or patchy erythma (4/20)  None
     72 hrs  Discrete or patchy erythma (2/20)  None
   Vehicle  48 hrs  Absent  None
     72 hrs  Absent  None
 Vehicle Group  TA  48 hrs  Absent  None
     72 hrs  Absent  None
   Vehicle  48 hrs  Absent  None
     72 hrs  Absent  None
Interpretation of results:
GHS criteria not met
Conclusions:
No responses in the substance-induced animals exceeded those of the vehicle-induced group animals. All responses were attributed to the primary irritative properties of the test article, and not to a sensitization response. Therefore, the substance is not considered to be a dermal sensitiser.

Executive summary:

A Guinea Pig Maximisation Test was undertaken to determine the sensitising potential of the substance when applied dermally. The study was conducted according to the OECD Guideline No. 406. Induction A consists of intradermal injections followed in seven days by Induction B topical application of the test article. The Challenge consists of a topical application performed 14 days following completion of the induction phases. No responses in the substance-induced animals exceeded those of the vehicle-induced group animals. All responses were attributed to the primary irritative properties of the test article, and not to a sensitization response. Therefore, the substance is not considered to be a dermal sensitiser. The substance is not categorized according to the Global Harmonized System (GHS) of Classification and Labeling of Chemicals.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Read-across to K2 study therefore K2 is the maximum Klimisch value.
Justification for type of information:
Read-across approach - see read-across justification in section 13.
Reason / purpose for cross-reference:
read-across source
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The in vivo study data were obtained in studies performed before any in vitro sensitization tests tests had been validated and accepted for regulatory purposes. Additionally, literature data demonstrates that an LLNA method is unreliable for surfactant substance, and may provide false positive results[1]. Therefore, an LLNA method is not deemed reliable for assessing the skin sensitisation of the substance.

[1]: Evaluating the sensitization potential of surfactants: Integrating data from the local lymph node assay, guinea pig maximization test, and in vitro methods in a weight-of-evidence approach. Ball et al. Regulatory Toxicology and Pharmacology 60 (2011) 389–400
Species:
guinea pig
Strain:
other: Pirbright-White
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: no data
- Weight at study initiation: 300 - 430 g
- Housing: max. 2 animals in one cage, Macrolon cages
- Diet: ad libitum; Ssniff-G (Alleindiät für Meerschweinchen), Ssniff Versuchstier-Diaten GmbH, 4770 Soest/Westfalen
- Water: ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 40-70
- Air changes (per hr): 10 per hour
- Photoperiod: 12 hours daily
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
DOSE RANGE FINDING STUDY:
epicutaneous: 5%, 20%, 50%, 100%
intradermal: 0.1%, 0.5%, 1%, 5%

MAIN STUDY
Induction
intradermal: 5%
epicutaneous: 20%

Challenge
epicutaneous: 5%
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
DOSE RANGE FINDING STUDY:
epicutaneous: 5%, 20%, 50%, 100%
intradermal: 0.1%, 0.5%, 1%, 5%

MAIN STUDY
Induction
intradermal: 5%
epicutaneous: 20%

Challenge
epicutaneous: 5%
No. of animals per dose:
Range finding:
6 animals (4 epicutaneous, 2 intradermal)

Main Study:
10 animals (test group)
5 animals (control group)
Details on study design:
The test was performed according to a modified version of the Magnusson-Kligman Guinea Pig Maximisation Test. This investigation was performed according to HLD test plan P 3/152, 3-rd revision as well as according to the recommended guidelines of the USA Interagency Regulatory
Liaison Group (IRLG, January, 1981).

RANGE FINDING TESTS:
Four animals were treated dermally in a preliminary study under occlusiv conditions (exposure period 24 h) with the following concentrations of the sample: epicutaneous: 5%, 20%, 50%. Reading 3 h p.a.
Two animals were treated intradermal: 0.1%, 0.5%, 1%, 5% (aqueous solution). Reading 24 h p.a.

MAIN STUDY
A. INDUCTION EXPOSURE
A.1 INTRADERMAL INJECTION – Performed on Test Day 1
Based on the pretest results, the test item concentration of 5% was selected for intradermal induction in the main study.
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made just within the boundaries of a 4 x 6 cm area in the clipped region as follows:

- Test groups:
1. 0.1 ml Complete Adjuvant (50 % v/v in water for injection) (Bactoadjuvant Completa H 37 Ra, Difco Laboratories, Detroit, Michigan)
2. 0.1 ml 5 % v/v test substance in Aqua dest.
3. 0.1 ml 5 % v/v test substance in Aqua dest. emulsified in 50 % Adjuvant

- Control group:
1. 0.1 ml Complete Adjuvant (50 % v/v in water for injection)
2. 0.1 ml aqua dest
3. 0.1 ml Complete Adjuvant (50 % v/v in aqua dest.)

A.2 EPIDERMAL INDUCTION - Performed one week after intradermal injection
- Volume: 0.5 ml
- Exposure period: 48 h
- Test groups: 10
- Control group: 5
- Site: the same site as for intradermal injection
- Frequency of applications: 1
- Concentrations: 20%
B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 2 weeks after induction
- Exposure period: 24 h
- Control group: aqua dest.
- Concentrations: 5 %
- Evaluation (hr after challenge): 48h, 72h
Positive control substance(s):
not specified
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no skin reaction
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no skin reaction.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
5 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no skin reaction
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 5 %. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no skin reaction.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no skin reaction
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no skin reaction.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no skin reaction
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no skin reaction.
Interpretation of results:
GHS criteria not met
Conclusions:
Since no allergic responses were observed in this MAXIMIZATION test, the test substance is not a dermal sensitizer.
Executive summary:

In a dermal sensitization study with the substance in water, young adult Pirbright-White guinea pigs (15 males;10 test and 5 control) were tested using the MAXIMIZATION test method equivalent to OECD Guideline 406.

The maximum compatible concentrations which led to slight irritation after intradermal and dermal application as well as the subirritative dose for the challenge application were determined in pretests. Water was used as vehicle during induction and challenge. Based on the results of the pretests, for the intradermal and epicutaneous induction exposure test substance concentrations of 5% and 20% were used, respectively. The test article concentration for the challenge application was 5%.

No allergic skin reactions were observed in test animals 48 and 72 hours after the challenge exposure. No findings were observed in control animals.

The sensitisation rate, i.e. the number of animals showing an allergic response expressed as a percentage of the total number of animals, was 0 %.

In this study, the substance is not a dermal sensitizer.

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

The substance gave a positive result in a reliable LLNA. The positive result in the LLNA is consistent with that obtained from a old, unreliable human volunteer study dosed at 10%, where a positive skin sensitisation response was given in 41 out of 200 cases (20.5% response rate). However there are a number of reasons why the validity of the LLNA result is open to question:

1. The chemical structure does not contain any obvious structural features which are normally associated with skin sensitization ie the presence or metabolic/non-metabolic formation of protein reactive functional groups.

 

2. The structurally related substance N,N,N-trimethyl-3-(undec-10-enoylamino)propan-1-aminium methyl sulfate (CAS 94313-91-4, EC 304-990-8) has been tested in a reliable Guinea Pig Maximisation Test (GPMT) and gave a negative response.

3. There is significant discussion in the toxicology literature regarding the validity of positive results obtained in the LLNA on irritant, surfactant substances. One of the more recent references (Ref 1, Ball et al 2011) notes that:

 

Since its introduction, there have been publications reporting apparent false positive results from the LLNA for certain groups of chemicals, including surfactants (Garcia et al., 2010; Kreiling et al., 2008; Woolhiser et al., 1998), the conclusion being that the LLNA may in some cases be misrepresenting the sensitizing potential of the tested substances. The basis for claims that the assay gives ‘false positives’ comes from contradictory data from experimental results obtained from other animal models, e.g. the GPMT, human data (hRIPT, worker/consumer experience), and the lack of structural alerts within the substance that are associated with sensitizing potential.

 

The paper also recommends that future in vivo testing of surfactants for sensitizing potentials should be done using one of the available guinea pig test guidelines to insure the highest relevance of the results to humans.

On the basis of the concerns listed above, the validity of the positive LLNA result on the substance is questionable. The human volunteer data which supports it is also old and unreliable. Therefore further skin sensitization testing in a guinea pig assay (GPMT) was undertaken to confirm or refute the validity of the LLNA result. The GPMT study gave a negative ie non-sensitising result.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance gave a positive skin sensitisation response in a reliable LLNA (three fold increase at a dose concentrations of 2% substance and above) and in an old, unreliable human volunteer study. The substance had been classified as a skin sensitiser, Category 1A based on this data. However, a number of uncertainties (discussed above) were identified and the validity of the positive LLNA result on the substance was considered questionable. Therefore further in vivo skin sensitization testing was undertaken to confirm or refute the validity of the LLNA result. In a reliable Guinea Pig Maximisation Test, no responses in the substance-induced animals exceeded those of the vehicle-induced group animals and so the substance is not considered to be a dermal sensitiser in this study. This result is considered to be more reliable than the LLNA result or the human volunteer data and is also consistent with the results of the GPMT study on the analogue substance. On this basis the substance is not classified.