6-sec-butylquinoline

Endpoint summary

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:

28 May to 21 July 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

April 29, 1993

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Novembre 2000 (draft)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- purtity: 99.9%
- Batch no.: 9000456328
- Expiry date: 22-JAAN-2004
- Storage condition: in the original container at room temperature (20±3°C), away from direct sunlight

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Remarks:

Recognized as the recommended test system

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands - B.V. Postbus 6174 - NL - 5960 AD Horst / The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: not available
- Age at acclimatization start: 7-12 weeks (beginning of acclimatization period)
- Weight at acclimatization start: 16.4 g - 22.2 g (beginning of acclimatization period)
- Housing: in groups of four in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz)
- Diet (e.g. ad libitum): ad libitum, pelleted standard Kliba 3433, batch no. 24/02 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst)
- Water (e.g. ad libitum): ad libitum, community tap water from Itingen
- Acclimation period: one week. Under test conditions after health examination. Only animals without any visible signs of ilness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Standard laboratory conditions, air conditioned with target ranges room temperature 22±3°C
- Humidity (%): relative humidity 30-70%
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light / 12 hour dark cycle

Vehicle:

other: Ethanol:water, 7:3 (v/v)

Concentration:

- Test item at 0.1, 1, 10, 50 and 100% (w/w) in ethanol:water, 7:3 (v/v)

No. of animals per dose:

- Four animals per dose and four animals for positive control

Details on study design:

TOPICAL APPLICATION
The test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with the test item at concentrations of 0.1%, 1%, 10%, 50% (w/w) in ethanol:water, 7:3 (v/v) and 100% (undiluted). The application volume, 25 microliter, was spread over the entire dorsal surface (diameter around 8 mm) of each 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) was purchased from Amersham International.
Five days after the first topical application, all mice were administered with 250 microliter of 79.77 microCi/ml 3HTdR (equal to 19.9 microCi 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 VETANARCOL (Veterinaria AG, Zürich) at 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 micrometer 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°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 1ml-aliquits of 5% tricholoroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegration per minute (DPM).

Positive control substance(s):

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

Statistics:

Mean values and standard deviations were calculated for body weights.

Positive control results:

Control item 5% (w/w): Stimulation Index = 2.2
Control item 10% (w/w): Stimulation Index = 2.0
Control item 25% (w/w): Stimulation Index = 6.7

Key result

Parameter:

SI

Value:

ca. 1

Test group / Remarks:

0.1% (w/w) test item

Key result

Parameter:

SI

Value:

ca. 1.1

Test group / Remarks:

1.0% (w/w) test item

Key result

Parameter:

SI

Value:

ca. 2.6

Test group / Remarks:

10% (w/w) test item

Key result

Parameter:

SI

Value:

ca. 15.6

Test group / Remarks:

50% (w/w) test item

Key result

Parameter:

EC3

Value:

ca. 11.2

Test group / Remarks:

EC3 therotically calculated with SI of group 10% (w/w) test item and 50% (w/w) test item

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The proliferation capacity of pooled lymph node cells was determined by the incorporation of 3HTdR measured on a beta-scintillation counter. A test item is regarded as a sensitizer if the exposure to at least one concentration resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.

EC3 CALCULATION
A statistical analysis was conducted for assessment of dose-response relationship. 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 proliferation activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately, if present, above and below the Stimulation Index value of 3 on the local lymph node assay dose response plot.

VIABILITY/MORTALITY
No deaths occurred during the study period with the exception of test group 100%, undiluted.

CLINICAL OBSERVATIONS:
During the study period, no symptoms of local toxicity at the ears of the animals and no systemic findings were observed in all animals with the exception of test group 100%, undiluted. About 22 hours after the first topical application all animals of the test group 100%, undiluted, showed moderate to severe sedation and decreased activity, ventral recumbency or hunched posture. Some of them showed moderately labored respiration (2 mice) and slightly ruffled fur (3 mice). For ethical reason all animals of test group 100%, undiluted were euthanized.

BODY WEIGHTS
The body weights of the animals, recorded at the start of acclimatization period and prior to necropsy, 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:

In the study Stimulation Index of 1.0, 1.1, 2.6 and 15.6 were determined with the test item at concentrations of 0.1%, 1%, 10% and 50% (w/w) in ethanol:water, 7:3 (v/v).
A test item is regarded as a sensitizer if the exposure to at least one concentration resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
Based on these criteria, the test item showed an allergenic potency when tested at concentration of 50% (w/w).
EC3 is the estimated concentration for stimulation index of 3. In this study EC3 of 11.2% (w/w) was theoretically calculated with stimulation indices of 2.6 and 15.6 at test item concentrations of 10% and 50% (w/w).
According to Basketter D. A. at al., 2002, the test item could be classified as a weak contact allergen (Class 3, EC3 value > 10-30%).
According to the low concentrations tested (0.1%, 1% and 10%) and corresponding stimulation index (1.0, 1.1 and 2.6) that are below 3 and the EC3 of 11.2% which is > 2% it is relevant to classify the test item in the sub-category 1B (skin sensitizing potential) regarding the CLP criteria. (Reference: Guidance on the Application of the CLP Criteria Version 4.0 – November 2013)
The test item was found to be systemically toxic when applied undiluted to the dorsal ear surface.

Executive summary:

In order to study a possible allergenic potential of the test item, five groups of four female mice each were treated with the test item at concentrations of 0.1%, 1%, 10%, 50% (w/w) in ethanol:water, 7:3 (v/v) and 100% (undiluted) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days.a control group of four mice was treated with the vehicle (ethanol:water, 7:3 (v/v)) only. Five days after the first topical application, the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-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 washed subsequently and incubated in trichloroacetic acid overnight the proliferation capacity of pooled lymph node cells was determined by the incorporation of³H-methyl thymidine measured in ab-scintillation counter.

No test-item related clinical signs were observed in all animals with the exception of test group 100%, undiluted.

About 22 hours after the first topical application all animals of the test group 100%, undiluted, showed moderate to severe sedation and decreased activity, ventral recumbency or hunched posture. Some of them showed moderately labored respiration (2 mice) and slightly ruffled fur (3 mice). For ethical reason all animals of test group 100%, undiluted were euthanized.

All treated animals survived the scheduled study period with exception of test group 100%, undiluted.

A test item is regarded as a sensitizer if the exposure to at least one concentration resulted in an incorporation of³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.

 

Results:

Test item 0.1% (w/w): Stimulation Index = 1.0

Test item 1.0% (w/w): Stimulation Index = 1.1

Test item 10% (w/w): Stimulation Index = 2.6

Test item 50% (w/w): Stimulation Index = 15.6

Conclusion: a clear dose-response relation was observed. EC3 of 11.2% (w/w) was theoretically calculated with stimulation indices of 2.6 and 15.6 at test item concentrations of 10% and 50% (w/w).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available