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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 31 May,2012 to 11 June,2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant with EU guideline.
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
The diet 4 RF 21 was used instead of 4 RF 18. No effect on results
Deviations:
yes
Remarks:
The diet 4 RF 21 was used instead of 4 RF 18. No effect on results
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia
Strain:CBA/JN
- Age at study initiation:6 to 8 weeks old
- Weight at study initiation:16 to 25 g
- Housing:1/cage during the study; up to 5 during acclimatisation. Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm and nesting material
- Diet :4 RF 21 (Mucedola ) Ad libitum throughout the study
- Water : Drinking water supplied to each cage via a water bottle Ad libitum.
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 2°C
- Humidity: 55% ± 15%
- Air changes : Approximately 15 to 20 air changes per hour
- Photoperiod: Artificial (fluorescent tubes), daily light/dark cycle of 12/12 hours

IN-LIFE DATES: From: To:
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary phase : 100, 50, 25, 10, 5% w/w
Main Phase : 10, 5, 2.5% w/w
No. of animals per dose:
Preliminary phase

Group Treatment Dose level Animal number
Number (% w/w)+ Females (odd)

1 Test item 100 1
Test item 50 3
Test item 25 5
Test item 10 7
Test item 5 9
Vehicle 0 11

+ = in terms of test item as supplied


MAIN PHASE

Group Treatment Dose level Animal number
Number (% w/w)+ Females (odd)

1 Vehicle 0 13-19
2 Test item 2.5 21-27
3 Test item 5 29-35
4 Test item 10 37-43
5 Positive control 25 45-51

+ = in terms of test item and positive control item as supplied





Details on study design:
RANGE FINDING TESTS:
- Irritation: A preliminary irritancy test was carried out to select three concentrations to be used in a main assay, according to the criteria described in the relevant guideline for this test. A main phase was then carried out to fully evaluate lymph node cell reaction.





Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.
Positive control results:
In the group treated with the positive control item, a stimulation index (SI) of 5.73 was calculated. As it was greater than 2, the test system was regarded as valid.
Key result
Parameter:
SI
Value:
ca. 2.48
Test group / Remarks:
Group n. 2 at a dose level of 2.5%
Key result
Parameter:
SI
Value:
ca. 2.85
Test group / Remarks:
Group n. 3 at a dose level of 5%
Key result
Parameter:
SI
Value:
ca. 4.04
Test group / Remarks:
Group n. 4 at a dose level of 10% w/w selected as highest dose on the basis of the preliminary study.
Cellular proliferation data / Observations:
Statistically significant and dose-related increases in cell proliferation of draining lymph nodes were observed in the three treatment groups, being the calculated stimulation indices (SI) 2.48, 2.85 and 4.04, respectively at low, mid- and high dose levels. A single animal of the negative control group (animal no. 91150019) showed an abnormally sized lymph node which caused a high response when compared to the group-mates and to the normal ranges observed in this experimental conditions. Therefore, although no reason for this abnormal result was found, these outlier data were excluded.

Preliminary test

Five concentrations (100, 50, 25, 10, 5% w/w) were selected to be used in the preliminary phase.

No toxicity signs (clinical signs or toxicologically relevant body weight losses) were observed at any of the tested concentrations.

The evaluation of visible reactions was as follows:

-Very slight to well defined erythema (scores of 1 and 2) was observed at the concentrations of 100 and 50% w/w;

-Very slight erythema (score of 1) was recorded at the concentration of 25% w/w;

-Desquamation and/or hardening of the treated ears were observed at the concentrations of 100, 50 and 25% w/w;

-No erythema was recorded at the concentrations of 10 and 5% w/w.

The evaluation of ear thickness indicated that the reaction was acceptable (increase of less than 25% compared to Day 1) at the concentrations of 10 and 5% w/w.

The evaluation of ear punch weight indicated that the reaction was acceptable (increased less than 25% with respect to the negative control) at the concentrations of 25, 10 and 5% w/w.

Main assay

Body weight decreases/reduced body weight gains observed in some animals from all treated groups were considered of low entity and/or incidental and, thus, not toxicologically relevant.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The results obtained in this study indicate that the test item may elicit a sensitisation response in mice following dermal exposure.
Executive summary:

The potential of the test item, PARAFFIN WAXES AND HYDROCARBON WAXES, CHLORO, SULFOCHLORINATED SAPONIFIED (C14-C17) SSP-SAMPLE 1, to cause skin sensitisation reactions following topical application to the skin of CBA/JN (CBA/J) mice, was assessed using the LLNA:BrdU-ELISA method, according to the OECD Guideline for testing of chemicals no. 429.

Preliminary phase

Five concentrations (100, 50, 25, 10, 5% w/w) in acetone:olive oil 4:1 (v/v) were selected to be used in the preliminary phase, in order to identify a non toxic and minimally irritant concentration and avoid false positive results. No signs of toxicity (toxicologically relevant clinical signs or body weight losses) were observed at the tested concentrations. According to the results of the irritation screening, the concentration judged as minimally irritant was 10% w/w.

Main assay

In the main assay, the test item was topically administrered at the concentrations of 10, 5 and 2.5% w/w, in acetone:olive oil 4:1 (v/v).

No mortality or clinical signs were recorded in any animal. Changes in body weight observed during the study were within the expected range for this strain and age of animals. Statistically significant and dose-related increases in cell proliferation of draining lymph nodes were observed in the three treatment groups, being the calculated stimulation indices (SI) 2.48, 2.85 and 4.04, respectively at low, mid- and high dose levels. The results obtained in this study indicate that the test item may elicit a sensitisation response in mice following dermal exposure. European Directives concerning the classification, packaging and labelling of dangerous substances (Council Regulation (EC) No. 1272/2008 and subsequent revisions) would indicate the following:

Classification: Category 1B

Signal word : Warning

Hazard statement: H317: May cause an allergic skin reaction

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

A possibility that the LLNA study is giving a false positive result exists, supported by the fact that with irritant substances this is not a very reliable test, even if Regulation recommend it always as a first choice.

The study has been performed on a dose-range where the irritating effect seems not being interferring with the test result, but the doubt remains.

We didn't performe an in vivo study for confirmation and assumed the classification as a conservative approach, taking also into account that the raw material too has been classified as sensitizer.

 


Migrated from Short description of key information:
The substance is sentizing according to the performed LLNA

Justification for selection of skin sensitisation endpoint:
The study is within validity criteria, in GLP, well described

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test item is considered to induce sensitisation when the SI for any single treatment dose group is ³ 1.6. Following the reported table:

Group

%

SI

1

0

1

2

2.5

2.48

3

5

2.85

4

10

4.04

5

25

5.73

and according to Reg. 286/2001 (II ATP CLP) a substance has to be classified as a sensitizer category 1B if the EC3 value > 2%. In this case the EC3 value (where SI = 3) is between concentrations of 5 -10%.

Therefore the proposed classification is H317, category 1B