Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-05-30 to 2015-05-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: Pre-test: 9 - 10 weeks Main study: 8 - 9 weeks
- Weight at study initiation: 17.8-20.9 g
- Housing: grouped by test groups, Makrolon Type II (pre-test) / III (main study), with wire mesh top
- Diet: ad libitum 2018C Teklad Global 18 % protein rodent diet
- Water: ad libitum tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
25, 50, 100 %
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: experiment was performed according to the recommendations given by OECD 429
- Irritation: 2 animals treated with 50 and 100 % on 3 consecutive days
- Lymph node proliferation response: no

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: a) exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index b) data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
- Animals were treated topically on the dorsal surface of each ear (25 μL/ear/day) on 3 consecutive days
- 5 days after first application 81.3 μCi/mL 3HTdR were injected
- 5 h after injection animals were sacrificed
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
Where appropriate, the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations validated statistical program R Script DecisionTree_2.Rnw was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and the Grubb’s test were used for detection of possible outliers (performed with validated statistical program R Script Outlier.Rnw).
However, both biological and statistical significance were considered together.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: - Vehicle control: 1.0 - 25 %: 1.45 - 50 %: 2.06 - 100 %: 6.49
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM per animal (2 lymph nodes) - Vehicle control: 1716.3 ± 358.7 - 25 %: 2481.9 ± 967.9 - 50 %: 3528.1 ± 1020.7 - 100 %: 11131.3 ± 7052.4

Any other information on results incl. tables

Viability / Mortality

No deaths occurred during the study period.

 

Clinical Signs

No signs of systemic toxicity were observed during the study period. From day 2 (1 hour after the second application) to day 5, the animals treated with a test item concentration of 100 % showed an erythema of the ear skin (score 1). Additionally the animals treated with the undiluted test item transiently showed reduced spontaneous activity on day 2 and burrowed themselves in the bedding (1 hour after the second application). Animals treated with 25 and 50 % test item concentration did not show any signs of local skin irritation or symptoms of toxicity.

 

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

 

Lymph Node Weights and Cell Counts

A statistically significant increase in lymph node cell counts was observed in the mid and high dose group in comparison to the vehicle control group. Additionally, a statistically significant increase in lymph node weight was observed in the high dose group in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell exceeded this threshold in the mid and high dose group (index of 1.75 and 2.65, respectively).

 

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not reached or exceeded in any of the treated groups.

 

Applicant's summary and conclusion

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
Migrated information
Conclusions:
The test item 1,4-Butanediol Divinyl Ether was found to be a skin sensitizer and an EC3 value of 60.6% (w/w) was derived.