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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:
2001
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Guideline study performed under GLP. The source and target substances must have an aldehyde group at the 1-carbon position and either a terminal (10-position) or an internal alkene (9-position or 8-position; with the terminal alkyl group no larger than an ethyl group and/or should not multiply substituted). The substance alkyl chain length of the substance should be more than 6 carbons in length and less than 14 carbons in length and fulfil the mono-alkene definition. The substance should not have any branched alkyl groups or side chains. The target and source share common structural elements in the same relative positions. The source and target have very similar physico-chemical properties and thus have similar expected toxicokinetic behaviour. The substances have similar in silico chemical reactivity predictions. This is observed within available in vivo toxicology testing where low level local and systemic toxicity is demonstrated and comparable between target and source. The substances therefore demonstrate chemical similarity. In the skin sensitisation endpoint, the target and source have common protein reactivity alert(s). The target and source therefore have common foreseen mode of action.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
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:
- Physical state: liquid
- Stability under test conditions: The solutions were used as soon as they were prepared to ensure the stability of the test material in solution.
- Storage condition of test material: In the original container in refrigerator (at 3 - 5 °C), away from direct sunlight

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Jibm(SPF)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Recognised animal supplier
- Age at study initiation: 7 - 12 weeks
- Weight at study initiation: 17.7 - 24.8 g
- Housing: In groups of four in Makrolon type-3 cages with standard softwood bedding
- Diet: standard pellet diet ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: 28-AUG-2001 to 12-SEP-2001

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5, 10, 25, 50 and 75%
No. of animals per dose:
4 females
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:

The test item in the main study was assayed at five consecutive concentrations that were selected by the sponsor.

TOPICAL APPLICATION
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5 %, 10 %, 25 %, 50% and 75% in acetone:olive oil, 4:1 (v/v). The application volume, 25uI, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

ADMINISTRATION OF 3H-METHYL THYMIDINE
3H-methyl thymidine (3HTdR) (specific activity, 2 Ci/mmol; concentration, 1 mCi/ml). Five days after the first topical application, all mice were administered with 250uL of 83.32 uCi/ml 3HTdR (equal to 20.83 uCi 3HTdR) by intravenous injection via a tail vein.

DETERMINATION OF INCORPORATED 3HTDR
Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of NARCOREN at a dose of at least 2 ml/kg body weight (equivalent to 320 mg sodium pentobarbitone/kg body weight). The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 um mesh size). After washing three times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 degrees C overnight for precipitation of macromolecules. The precipitates were then resuspended in 5% trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5 %trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive
disintegrations per minute (DPM).
Positive control substance(s):
mercaptobenzothiazole (CAS No 149-30-4)
Statistics:
The mean values and standard deviations were calculated in the body weight tables. 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.l. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:
2-MERCAPTO-BENZOTHIAZOLE showed an allergenic potency when tested at concentrations of 10 % and 25 %.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see table 1 below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see table 1 below

Any other information on results incl. tables

Table 1. Individual results

Test concentration % Group Measurement dpm   Calculation   Result - S.I.
dpm - BG Number of lymph nodes dpm per lymph node
- BG I 4 - - - -
- BGII 4 - - - -
- CG I 4146 4142 8 518 -
5 TG 2 7028 7024 8 878 1.7
10 TG 3 21816 21812 8 2727 5.3
25 TG 4 31132 31128 8 3891 7.5
50 TG 5 36016 36012 8 4502 8.7
75 TG 6 36335 36331 8 4541 8.8

BG = Background (1 mL 5% trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.l. = Stimulation Index

EC3 was calculated as 6.8%

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of this study the EC3 of the test substance has been calculated as 6.8% and is considered to be weakly sensitising.
Executive summary:

The study was performed to GLP and the method followed OECD 429 to assess the skin sensitisation potential of the test material in the CBA/Jibm(SPF) strain mouse following topical application to the dorsal surface of the ear. The test item in the main study was assayed at five consecutive concentrations that were selected by the sponsor. Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5 %, 10 %, 25 %, 50% and 75% in acetone:olive oil, 4:1 (v/v). The application volume, 25 uL, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). Five days after the first topical application, all mice were administered with 250 uL of 83.32 uCi/ml 3HTdR (equal to 20.83 uCi 3HTdR) by intravenous injection via a tail vein. The mice were observed twice daily and any clinical signs of toxicity, if present, were also recorded. The bodyweight was recorded at the start of acclimatisation and prior to necropsy. No irritation or signs of systemic toxicity were observed in the animals examined. Body weights were within the range seen for controls over the study period. Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 10, 25, 50 and 75% were 7024, 21812, 31128, 36012 and 36331 DPM, respectively. The mean DPM/animal value for the vehicle control group was 4142 DPM. The SI values calculated for the substance concentrations 5, 10, 25, 50 and 75% were 1.7, 5.3, 7.5, 8.7 and 8.8, respectively. Under the conditions of this study the EC3 of the test substance has been calculated as 6.8% and is considered to be weakly sensitising.