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Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 09 may 2007 and 15 May 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
No
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaBkl)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: B & K Universal Ltd, hull, UK
- Age at study initiation: 8 to 12 weeks old.
- Weight at study initiation: 15 to 23 g.
- Housing: Individually housed in suspended solid-floor plypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): Free access to food (Certified Rat and Mouse Diet)
- Water (e.g. ad libitum): Free access to mains tap water
- Acclimation period: At least five days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approx 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light (06.00 to 18.00) and 12 hours darkness

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
Groups of four mice were treated with the test material at concentrations of 5%, 10% or 25% w/w in dimethyl formamide.
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
Information available suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration.
- Compound solubility: For the purpose of the study, the test material was freshly prepared in dimethyl formamide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. 25%: 1g of 50% dilution made up to 2g



MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitiser".



TREATMENT PREPARATION AND ADMINISTRATION:
Preparation:
For the purpose of the study, the test material was freshly prepared in dimethyl formamide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.

Administration:
The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material 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):
other: alpha-Hexylcinnamaldehyde, Tech, 85%
Statistics:
None provided.

Results and discussion

Positive control results:
Three groups, each of five animals, were treated with 50 µl (25 µl per ear) of alpha- Hexylcinnamaldehyde, Tech, 85% as a solution in dimethyl formamide at concentrations of 5%, 10% and 25% v/v. A further control group of five animals was treated with dimethyl formamide alone.

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:

Concentration (% v/v) in dimethyl formamide: 5
Stimulation Index: 2.81
Result: Negative

Concentration (% v/v) in dimethyl formamide: 10
Stimulation Index: 4.20
Result: Positive

Concentration (% v/v) in dimethyl formamide: 25
Stimulation Index: 13.15
Result: Positive

alpha-Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Concentration (% w/w) in dimethyl formamide Vehicle: na 5: S.I of 1.86 10: S.I of 1.68 25: S.I of 1.69
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Concentration (% w/w) in dimethyl formamide Vehicle: 3962.96 dpm 5: 7376.88 dpm 10: 6653.06 dpm 25: 6695.16

Any other information on results incl. tables

The radioactive disintegrations per minute per lymph node and the stimulation index are given in Table 1.

A stimulation index of less than 3 was recorded for the three concentrations of the test material (5%, 10% and 25% w/w in dimethyl formamide).

Table1               Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration

(% w/w) in
dimethyl formamide

dpm

dpm/Nodea

Stimulation Indexb

Result

Vehicle

3962.96

496.37

na

na

5

7376.88

922.11

1.86

Negative

10

6653.06

831.63

1.68

Negative

25

6695.16

836.90

1.69

Negative

dpm=    Disintegrations per minute

a=          Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number             of lymph nodes)

b=         Stimulation Index of 3.0 or greater indicates a positive result

na =      Not applicable

Clinical Observations and Mortality Data

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Purple/pink-coloured staining to the ears and fur was noted in all test animals post-dose on Day 1 and for the remainder of the test.

 Bodyweight

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

The test material was considered to be a non‑sensitiser under the conditions of the test.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
A stimulation index of less than 3 was recorded for the three concentrations of the test material (5%, 10% and 25% w/w in dimethyl formamide).

The test material was considered to be a non‑sensitiser under the conditions of the test.
Executive summary:

A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

§        OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted)

§        Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/73/EC

Three groups, each of four animals, were treated with 50 µl (25 µl per ear) of the test material as a solution in dimethyl formamide at concentrations of 5%, 10% or 25% w/w. A further group of four animals was treated with dimethyl formamide alone.

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:

Concentration (% w/w) in dimethyl formamide

Stimulation Index

Result

5

1.86

Negative

10

1.68

Negative

25

1.69

Negative

The test material was considered to be a non‑sensitiser under the conditions of the test.