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Description of key information

The substance was tested in a reliable GPMT using ten test and five control animals at dose concentrations as follows: Induction intradermal: 5% epicutaneous: 20% Challenge epicutaneous: 5%. The test material gave a negative response. A structurally related substance (methyl trimethyl-3-[(1-oxododecyl)amino]propylammonium sulphate; CAS 10595-49-0) gave a positive result in a LLNA. The latter result was supported by the result of an old, unreliable human study also conducted on the structural analogue. However there are concerns raised in the toxicology literature (e.g. reference 1) regarding the validity of positive results obtained in the LLNA on irritant, surfactant substances. It is thought that the LLNA tends to overestimate the sensitisation potential of surfactants. For this reason the positive LLNA result on the structurally related substance has been disregarded, together with the unreliable human result, and the classification proposal is based on the GPMT conducted on the substance itself.

Ref 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


Short description of key information:
The substance was negative in a Guinea Pig Maximisation Test. A structurally related substance gave a positive result in a reliable LLNA however there are concerns regarding the validity of this result. The structurally related substance also gave a positive result in an unreliable human study.

Justification for selection of skin sensitisation endpoint:
Two reliable studies are available, one on the substance and the other on a structurally related substance. The selected study is a GPMT on the substance itself.

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:
other information
Study period:
12/12/2007 to 25/12/2007
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
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

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:
sensitising
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 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:
experimental study
Adequacy of study:
key study
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
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
Reading:
1st reading
Hours after challenge:
48
Group:
test group
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.
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
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:
not sensitising
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)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance gave a negative result response in a reliable Guinea Pig Maximisation Test. Therefore there is no justification for classification of the substance.