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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
16 May 2012 - 29 May 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed in compliance with GLP and in accordance with the standardised guidelines OECD 429, and EU Method B.42. The study was performed to a high standard sufficient to assess the quality of the presented results. Since the study was conducted on the read across substance, dioctyltin oxide, it has been assigned a reliability score of 2.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
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: solid (unspecified)
Details on test material:
- Name of test material (as cited in study report): Di-n-octyltin oxide
- Physical state: white solid
- Storage conditions: room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: Individually
- Diet: ad libitum
- Water: Mains tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 ºC
- Humidity (%): 30 - 70 %
- Air changes (per hr): Approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours darkness

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
5, 10 or 25 % w/w
No. of animals per dose:
Animals were tested in groups of four.
Details on study design:
RANGE FINDING TESTS: a preliminary screening test was performed on one mouse. The mouse was treated by daily application of 25 µL of the test material at a maximum attainable concentration of 25 % w/w in propylene glycol, to the dorsal surface of each ear for three consecutive days. The mouse was observed twice daily on days 1, 2 and 3 and once daily on days 4, 5 and 6. Local skin irritation was scored daily according to the scale outlined in the field “Any other information on materials and methods incl. tables”. Any signs of toxicity, if present, were also recorded.
The thickness of each ear was measured using an Oditest micrometer pre-dose on day 1, post dose on day 3 and again on day 6. Any changes in ear thickness were noted.
- Compound solubility: 25 % w/w was selected as the highest dose investigated in the main test, reflecting the maximum attainable concentration of test material in vehicle. The vehicle was chosen as it produced the highest concentration of test material that was suitable for dosing.


TREATMENT PREPARATION AND ADMINISTRATION: Main test
Approximately 25 µL of 5 %, 10 % or 25 % w/w preparation of the test material in propylene glycol was applied using an automatic micropipette, to the dorsal surface of each ear. A vehicle control group was similarly treated using propylene glycol alone. The procedure was repeated daily for 3 consecutive days.

Five days after the first application, all the animals were injected, via the tail vein, with approximately 250 µL of phosphate buffered saline (PBS) containing approximately 80 µCi/mL of 3H-methyl thymidine with a specific activity of 2.0 Ci/mmol (total dose 20 µCi per mouse). Approximately 5 hours later, the animals were humanely killed by carbon dioxide asphyxiation. The draining aurcular lymph nodes were removed from each animal and, together with the nodes from the other animals in the group, were placed in a container of PBS.

A single cell suspension was prepared by mechanical disaggregation of lymph nodes through a 200-mesh stainless steel gauze. The cell suspensions were then washed twice times by centrifugation with approximately 10 mL of PBS. The cells suspensions were pelleted again and resuspended in approximately 3 mL of 5 % w/v trichloroacetic acid (TCA) , and precipitated overngiht (approximately 18 hours) at 4 °C. The samples were again pelleted by centrifugation and the supernatant was discarded. The cells were then resuspended in approximately 1 mL of TCA. The lymph node suspensions were transferred to scintillation vials and 10 mL of scintillant (Optiphase) was added prior to β-scintillation counting using a Packard Tri-Carb 2500TR Liquid Scintillation counter.

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE:
The results are expressed as disintegrations per minute (dpm) value per lymph node for each group. The activity of each test group is then divided by the activity of the vehicle control group to give a test:control ratio for each concentration. The criterion for a positive response is that one or more concentrations of the test material should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group. A test material which does not fulfil the above criterion is designated as unlikely to be a sensitiser.
Positive control substance(s):
other: phenylacetaldehyde (90 %) as a solution in propylene glycol at a concentration of 2.5 % v/v

Results and discussion

Positive control results:
The application of phenylacetaldehyde at concentrations of 2.5 % v/v in propylene glycol resulted in a greater than 3-fold increase in isotope incorporation. Therefore, phenylacetaldehyde was shown to be a skin sensitiser, confirming the validity of the protocol used for the study.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
The stimulation index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, is presented in table 2 in 'Any other information on results incl. tables'. The application of the test substance at concentrations of 5 %, 10 % and 25 % w/w in propylene glycol resulted in an isotope incorporation which was less than 3-fold at all three concentrations. Consequently, the test material was designated as unlikely to be a sensitiser under the conditions of the test.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
The pooled disintegrations per minute, disintegrations per minute per lymph node and stimulation index are presented in table 2 in 'Any other information on results incl. tables'. The test material is designated as unlikely to be a sensitiser under the conditions of the test.

Any other information on results incl. tables

Table 2: Disintegrations per Minute, Disintegrations per Minute/Node and Stimulation Index

Concentration (% w/w) in vehicle

dpm

dpm/node*

stimulation index #

Result

Vehicle

7746.32

968.29

na

na

5

7152.44

894.06

0.92

negative

10

6866.45

858.31

0.89

negative

25

13714.73

1714.34

1.77

negative

dpm = Disintegrations per minute

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

# Stimulation index of 3.0 or greater indicates a positive result

na = not applicable

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the test, the isotope concentration was less than three-fold at all test concentrations and therefore, the test material is considered to be unlikely to be a skin sensitiser. The study is considered to be reliable, relevant and adequate for risk assessment and classification and labelling purposes.
Executive summary:

The skin sensitisation potential of the test material was determined in accordance with the standardised guidelines OECD 429 and EU Method B.42 using the mouse Local Lymph Node Assay. The test material was applied as a 5 %, 10 % or 25 % w/w preparation in propylene glycol. The positive control was shown to have the capacity to cause skin sensitisation confirming the validity of the protocol used for this study. The isotope concentration induced by the test material was less than three-fold at all test concentrations and therefore the test material is considered to be unlikely to be a skin sensitiser.