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Toxicological information

Skin sensitisation

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Administrative data

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:
other: GLP study carried out in compliance with an internationally recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Lot No: K1270019
Purity: 99.9%
Storage conditions: room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: reputable supplier
- Age at study initiation: approximately eight to twelve weeks
- Weight at study initiation: 18.2 to 21.9 g
- Housing: solid bottomed polycarbonate cages with a stainless steel mesh lid.
- Diet: free access to standard rodent diet
- Water: potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours continuous dark

IN-LIFE DATES: From: To: 6 March to 25 March 2013

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25 and 50% v/v in Acetone:olive oil (4:1 v/v)
No. of animals per dose:
Two
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: formed a clear solution at 50% v/v in acetone:olive oil (4:1 v/v
- Irritation: vehicle trial indicated that the test material was suitable for dosing as supplied. Slight erythema was observed on the ears of one mouse treated with neat substance on Day 3. No erythma was observed on any animal at a concentration of 50%.
- Lymph node proliferation response: not measured

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Method: animals were assigned without conscious bias
- Criteria used to consider a positive response: results for each treatment group were expressed as the Stimulation Index (SI). This was derived by dividing the mean dpm/mouse for each treated group and the positive control group by the mean dpm/mouse in the vehicle control group.
The positive control group is expected to give an SI of 3 or more to demonstrate the validity of the study.


TREATMENT PREPARATION AND ADMINISTRATION: The results of the preliminary investigations indicated that 50% v/v was a suitable high concentration for administration in the main phase of the study. Two female mice (per concentration) received a daily application of 25µL of appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette and was spread over the entire dorsal surface of the ear using the tip of the pipette.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Hexyl Cinnamic aldehyde 25% v/v:
44025.60 dpm, 8 lymph nodes per animal, dpm/node 5503.20, Stimulation index 6.4

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 10% v/v 0.7 25% v/v 1.1 50% v/v 0.4
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 10% v/v 4951.40 25% v/v 7661.10 50% v/v 2860.50

Any other information on results incl. tables

Main phase

There were no deaths and no signs of ill health or toxicity observed during this study. Greasy fur on the head was noted following each dosing occasion, this was related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance.

No signs of dermal irritation were seen on the ear during the study.

There was no indication of an effect of treatment on bodyweight gain. A loss in bodyweight was noted for all mice in the 50% treatment group and one mouse from in the positive control group over the study period.

Results of preliminary investigation

Mortality and clinical signs

Substance as supplied:

Greasy fur on the head was noted following dosing on days 1 and 2. At the pre-dose check on Day 3 signs of toxicity were noted in the both females, these included underactivity, hunched posture, fast breathing, partially closed eyelids, tilted trunk and irregular breathing. Approximately 80 minutes later one female died and as this level was clearly unsuitable for administration on the main study the remaining animal was sacrificed.

 

50% v/v:

Greasy fur on the head was noted following each dosing occasion, this was related to unoccluded dermal administration of a liquid formulation/vehicle and not an effect of the test substance. No deaths or signs of toxicity were seen throughout the study.

Dermal reactions

Substance as supplied:

Slight erythema was observed on the ears of one mouse on Day 3.

50% v/v:

No erythema was observed on the ears of either mouse on Days 1 to 6.

Bodyweight

A loss in bodyweight was noted for both mice dosed at 50% v/v over the study period On the basis of the results from the preliminary investigation, 50% v/v was considered a suitable high concentration for administration in the main phase of the study.

Group dpm/node and Stimulation Index

 

Group

Concentration

dpm

Number of lymph nodes per animal

dpm/node

Stimulation Index†

Result

+ = positive

- = negative

 

 

 

 

 

 

 

3

AOO

6903.40

8.0

862.93

n/a

n/a

4

10% v/v

4951.40

8.0

618.93

0.7

-

5

25% v/v

7661.10

8.0

957.64

1.1

-

6

50% v/v

2860.50

8.0

357.56

0.4

-

7

HCA 25% v/v

44025.60

8.0

5503.20

6.4

+

 

 

 

 

 

 

 

             Stimulation Index of 3 or more indicates a positive result

dpm       Disintegrations per minute less background count of 37.10

AOO      Acetone:olive oil (4:1 v/v) (vehicle control)

HCA      Hexyl cinnamic aldehyde (positive control)

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
DMI is not regarded as a potential skin sensitizer.