1,4-Dioxaspiro[4.5]decane, 8-[4-(4-methylphenyl)-3-cyclohexen-1-yl]-

Endpoint summary

Description of key information

Based in the results of a Local Lymph Node Assay (LLNA) according to OECD guideline 429, an EC3 value of 3.6 % (w/v) was derived, therefore the item was regarded as a skin sensitizer (category 1B) (reference 7.4.1-1).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 14, 2003 - October 24, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

April 24, 2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands B.V. Postbus 6174 NL - 5960 AD Horst
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16.5 - 24.2 g
- Housing: Groups of four mice in Makroion type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz)
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From day 1 to day 5

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Pretest for signs of irritation: 1, 2.5, 5, and 10 % (w/v)
Main test: 2.5, 5 and 10 % (w/v)

No. of animals per dose:

Pretest: 2 females
Main test: 4 females per group (3 per test group, 1 per control group) total 16 females

Details on study design:

RANGE FINDING TESTS
To determine the highest non-irritant test concentration or the highest technically applicable concentration, a pretest was performed in two mice with test item concentrations of 1, 2.5, 5 and 10 % (w/v) on one ear each. No irritation effects were observed at these concentrations after a single application. 10 % (w/v) was the highest technically achievable concentration in the chosen vehicle.

MAIN STUDY
- Name of test method: OECD TG 429
- Criteria used to consider a positive response: 3-fold greater response at one concentration in SI than control animals

TREATMENT PREPARATION AND ADMINISTRATION
Each test group was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with test item concentrations of 2.5, 5 and 10 % (w/v) in acetone:olive oil, 4:1 (v/v). The application volume of 25 μL 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 250 μL of 80.81 μCi/mL 3H-methyl thymidine (3HTdR) by intravenous injection via a tail vein. Prior to each treatment the ear thickness was measured. Approximately five hours after treatment with 3HTdR all mice were euthanized and draining lymph nodes were excised and pooled for each experimental group (8 nodes per group). After further preparation of the samples the level of 3HTdR incorporation was measured on a ß-scintillation counter.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Mean values and standard deviations were calculated in the body weight tables. A statistical analysis was conducted for assessment of the dose-response relationship, and 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.

Positive control results:

Conc. (%, w/v) SI
5 1.5
10 3.2
25 6.9

Key result

Parameter:

SI

Value:

3.3

Test group / Remarks:

2.5%

Key result

Parameter:

SI

Value:

2.6

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

2.3

Test group / Remarks:

10%

Key result

Parameter:

EC3

Remarks:

% (w/v)

Value:

3.6

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
control group: 283 dpm per lymph node
2.5%: 933 dpm per lymph node
5%: 748 dpm per lymph node
10%: 645 dpm per lymph node

EC3 CALCULATION
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 priliferative 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.

CLINICAL OBSERVATIONS:
No deaths occured during the study period. No test-item related clinical signs were observed in any animals of the control group, group 2 (2.5%) or group 3 (5%). On the third application day, a slight ear swelling was observed at both dosing sites in all mice of group 4 (10%), persisting for two days.

BODY WEIGHTS
The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

From this study according to OECD guideline 429, an EC3 value of 3.6 % (w/v) was derived, therefore the item was regarded as a skin sensitizer.

Executive summary:

In order to study a possible contact allergenic potential of the test item, three groups each of four female mice were treated daily with the test item at concentrations of 2.5%, 5% and 10% (w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days according to OECD guideline 429. 10 % was the highest technically achievable concentration in the vehicle. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a β-scintillation counter. No test item-related clinical signs were observed in any animals of the control group, Group 2 (2.5 %) or Group 3 (5 %). On the third application day, a slight ear swelling was observed at both dosing sites in all mice of Group 4 (10 %), persisting for two days. All treated animals survived the scheduled study period. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study, stimulation indices of 3.3, 2.6 and 2.3 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v). An EC3 value of 3.6 % (w/v) was derived, therefore the test item was regarded as a skin sensitiser. However, the results obtained showed a clear negative dose response, demonstrated by decreasing S.I. values. As no significant test item related findings were observed, it might be considered that the effect obtained is based on local cytotoxicity to the Langerhans cells as the immuno targets or a limited penetration rate with higher test item concentrations.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In vivo skin sensitisation, key study

In order to study a possible contact allergenic potential of the test item, three groups each of four female mice were treated daily with the test item at concentrations of 2.5%, 5% and 10% (w/v) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days according to OECD guideline 429. 10 % was the highest technically achievable concentration in the vehicle. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a β-scintillation counter. No test item-related clinical signs were observed in any animals of the control group, Group 2 (2.5 %) or Group 3 (5 %). On the third application day, a slight ear swelling was observed at both dosing sites in all mice of Group 4 (10 %), persisting for two days. All treated animals survived the scheduled study period. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study, stimulation indices of 3.3, 2.6 and 2.3 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in acetone:olive oil, 4:1 (v/v). An EC3 value of 3.6 % (w/v) was derived, therefore the test item was regarded as a skin sensitiser. However, the results obtained showed a clear negative dose response, demonstrated by decreasing S.I. values. As no significant test item related findings were observed, it might be considered that the effect obtained is based on local cytotoxicity to the Langerhans cells as the immuno targets or a limited penetration rate with higher test item concentrations.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the available data, the test item is classified as skin sensitiser cat. 1B and labelled with H317 (May cause allergic reactions) according to Regulation (EC) No 1272/2008 (CLP), as amended for fifteenth time in Regulation (EU) No 2020/217.