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Description of key information

In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, an EC3 value (the estimated test item concentration that will give a SI =3) of 2.4% was calculated for the analogue substance. The considered substance is considered to be a skin sensitiser and should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

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:
September 2015 to january 2016
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)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 2008, including most recent amendments
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Young adult animals (approx. 10 weeks old)
- Weight at study initiation: 18.4 - 24.9 g
- Housing: Animals were group housed in labeled Makrolon cages containing sterilised sawdust as bedding material. Paper and shelters were supplied as cage-enrichment. On Day 6, the animals were group housed in Makrolon cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 30 September 2015 to 26 October 2015
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 2, 5, 10% w/w test item
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. Initially, two test item concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
The test system, procedures and techniques were identical as those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (5% and 10%) at a later stage.

MAIN STUDY
- Name of test method: Local Lymph Node Assay
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
The EC3 value (the estimated test item concentration that will give a SI =3) was determined, using linear interpolation.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test item preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions.
Rationale for vehicle:The vehicle was selected on the basis of maximizing the solubility using the test item data provided by the Sponsor and trial preparation results performed at WIL Research Europe. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol and dimethylsulfoxide. There was no information available regarding the solubility or stability in vehicle.


Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing) according to a numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy for gross macroscopic examination was performed according to study plan.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.
Positive control results:
The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.
Parameter:
SI
Value:
2.5
Test group / Remarks:
2%
Parameter:
SI
Value:
6.4
Test group / Remarks:
5%
Parameter:
SI
Value:
10.1
Test group / Remarks:
10%
Key result
Parameter:
EC3
Value:
2.4
Cellular proliferation data / Observations:
Pre-screen Test
At 25% and 50% all animals were found dead on Day 2. At 5% and 10% no systemic toxicity was observed. Very slight to well-defined erythema was noted for the animals treated at 5% and 10%. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.
Based on these results, the highest test item concentration selected for the main study was a 10% concentration.

Skin Reactions / Irritation
Very slight erythema was noted for some animals treated at 5% and all animals treated at 10% between Days 2 and 4. This was considered not to have a toxicologically significant effect on the activity of the nodes.

Systemic Toxicity
No mortality occurred and body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
Piloerection was noted for some animals treated at 5% and all animals treated at 10% on Day 3. No further signs of systemic toxicity were observed in the animals of the main study.

Macroscopic Examination of the Auricular Lymph Nodes and Surrounding Area
All auricular lymph nodes of the animals of the animals treated at 2% and control group were considered normal in size. The lymph nodes of two animals treated at 5% and one or both nodes of all animals treated at 10%, appeared larger in size.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values
Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 1717, 4456 and 7008 DPM, respectively. The mean DPM/animal value for the vehicle control group was 697 DPM. The SI values calculated for the item concentrations 2, 5 and 10% were 2.5, 6.4 and 10.1, respectively.

The EC3 value for the 6 monthly HCA reliability check (positive control) was 10% which is in the acceptable range of 4.8% and 19.5%.
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, an EC3 value (the estimated test item concentration that will give a SI =3) of 2.4% was calculated. Tetrabutylphosphonium Bromide was considered to be a skin sensitiser and should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.
Executive summary:

An LLNA skin sensitisation study with tetrabutylphosphonium Bromide was performed according to OECD/EC test guidelines and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 2%, 5% and 10% w/w.

Very slight erythema was noted for some animals treated at 5% and all animals treated at 10% between Days 2 and 4. This was considered not to have a toxicologically significant effect on the activity of the nodes. No mortality occurred and body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Piloerection was noted for some animals treated at 5% and all animals treated at 10% on Day 3. No further signs of systemic toxicity were observed in the animals of the main study. The lymph nodes of two animals treated at 5% and one or both nodes of all animals treated at 10%, appeared larger in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 1717, 4456 and 7008 DPM, respectively. The mean DPM/animal value for the vehicle control group was 697 DPM. The SI values calculated for the item concentrations 2, 5 and 10% were 2.5, 6.4 and 10.1, respectively.

Since some animals in the middle and highest dose groups showed signs of systemic toxicity it cannot be excluded that this influenced the activity of the lymph nodes. However, since the test item elicits a SI ≥ 3 and the data show a clear dose-response, it was concluded that based on these results the test item should be considered as a skin sensitizer.

An EC3 value (the estimated test item concentration that will give a SI =3) of 2.4% was calculated.

Reliable negative and positive controls were included.

Based on these results:

- according to the recommendations made in the test guidelines (including all amendments), Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt would be regarded as skin sensitizer. - according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments), Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt should be classified as skin sensitizer (Category 1B).

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Tetrabutylphosphonium Bromide; CYPHOS® 442 Phosphonium Salt should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

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

An LLNA skin sensitisation study with the analogue substance was performed according to OECD/EC test guidelines and GLP principles. Based on the results of a pre-screen test, the test concentrations were selected at 2%, 5% and 10% w/w.

Very slight erythema was noted for some animals treated at 5% and all animals treated at 10% between Days 2 and 4. This was considered not to have a toxicologically significant effect on the activity of the nodes.No mortality occurred and body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Piloerection was noted for some animals treated at 5% and all animals treated at 10% on Day 3. No further signs of systemic toxicity were observed in the animals of the main study. The lymph nodes of two animals treated at 5% and one or both nodes of all animals treated at 10%, appeared larger in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and 10% were 1717, 4456 and 7008 DPM, respectively. The mean DPM/animal value for the vehicle control group was 697 DPM. The SI values calculated for the item concentrations 2, 5 and 10% were 2.5, 6.4 and 10.1, respectively.

Since some animals in the middle and highest dose groups showed signs of systemic toxicity it cannot be excluded that this influenced the activity of the lymph nodes. However, since the test item elicits a SI ≥ 3 and the data show a clear dose-response, it was concluded that based on these results the test item should be considered as a skin sensitizer.

An EC3 value (the estimated test item concentration that will give a SI =3) of 2.4% was calculated.

Reliable negative and positive controls were included.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

According to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments), the substance should be classified as skin sensitizer (Category 1B).

According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), the substance should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.