Reaction mass of 1-(3,3-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one and 1-(5,5-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one

Endpoint summary

Description of key information

Skin sensitisation (OECD TG 429): sensitising; EC3 and NOEC are 3% and 1%, respectively

HRIPT test: at 5%: not sensitising.

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:

04 April 2001, 11 April 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 2 as it has been conducted according to OECD Test Guideline 429 (Draft Nov. 2000) using the Skin Sensitisation: Local Lymph Node Assay method, without GLP.

Justification for type of information:

The LLNA study is justified because the test was carried out before the availability of in vitro sensitisation methods

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Draft November 2000

Deviations:

no

GLP compliance:

no

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands B.V., Postbus 6174, NL-5960 AD Horst, The Netherlands
- Age at study initiation: 6 - 8 weeks (beginning of acclimatization period)
- Weight at study initiation: 15.0 - 20.9 g (beginning of acclimatization period)
- Housing: In groups of four in Makrolon type-3 cages with standard softwoord bedding.
- Diet: Free access to pelleted standard Kliba 3433, batch no. 06/00 mouse maintenance diet (Provimi Kliba AG, CH-4132 Muttenz)
- Water: Free access to community tap water
- Acclimation period: Under test conditions after health examination, for 7 days.

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

Vehicle:

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

Concentration:

Undiluted test item (100%) or the test item at concentrations of 0.1%, 1% or 10% v/v in vehicle.

No. of animals per dose:

Groups of four mice were treated.

Details on study design:

The test item in the main study was assayed at four consecutive concentrations.
TREATMENT PROCEDURES:
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 0.1 %, 1 %, 1 0 % and 100 % (undiluted) in acetone:olive oil, 4:1 (v/v). The application volume, 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). 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 (Amersham product code no. TRA 31 0; specific activity, 2 Ci/mmol; concentration, 1 mCi/mL). Five days after the first topical application, all mice were administered with 250 µL of 84.78 µCi/mi 3HTdR (equal to 21.2 µCi 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 (Rhone Merieux GmbH, D-88471 Laupheim) 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 except for groups 4 and 5 where only 7 lymph nodes were found). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm 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 β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5% trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

OBSERVATIONS:
Mortality / Viability: Twice daily from acclimatization to the termination of in-life phase.
Body weights: At acclimatization start and prior to necropsy.
Clinical signs (local / systemic): Daily from acclimatization start to the termination of in-life phase. Especially the treatment sites were recorded carefully.

Positive control substance(s):

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

Statistics:

Not performed.

Positive control results:

The positive control item, hexyl cinnamic aldehyde, gave a Stimulation Index of 3.7 and 7.0 when tested at a concentration of 10 and 25 % v/v, respectively, in acetone/olive oil 4:1.

Key result

Parameter:

EC3

Remarks:

%

Value:

3

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 0.1, 1, 10 and 100% were 0.9, 0.9, 10.4 and 17.5, respectively.

Key result

Parameter:

other: NOEC

Remarks:

%

Value:

1

Interpretation of results:

other: sensitising.

Remarks:

According to Regulation (EC) No. 1272/2008.

Conclusions:

The SI values calculated for the substance concentrations 0.1, 1, 10 and 100% were 0.9, 0.9, 10.4 and 17.5, respectively. These results show that the test substance could elicit a SI ≥ 3. An EC3 has been derived resulted in an EC3 of 3%. A NOAEC of 1% is derived. The test substance was considered to be a sensitiser under the conditions of the test.

Executive summary:

The skin sensitisation potential of the substance has been tested according to OECD TG 429: Local Lymph Node Assay" method (draft November 2000), non-GLP. At 0.1, 1, 10 and 100% the substance showed SI values of 0.9, 0.9, 10.4 and 17.5, respectively. Reliable negative and positive controls were included.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. These results show that the test substance could elicit a SI ≥ 3.

An EC3 has been derived resulted in an EC3 of 3%. A NOEC of 1% is derived.

Based on the results, the substance was considered to be a sensitiser and should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction

according to Regulation (EC) No. 1272/2008 and GHS.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

 The starting value for the risk characterization is based on the LLNA because the skin sensitisation value in ug/cm2 is the more conservative value compared to the HRIPT:

The NOEC from the LLNAof 1% is converted to 232 ug/cm2 (based on an application volume of 25μL, an application area of 1 cm²and the relative density of the substance (0.926g/mL OR when using the ECHA guidance 1*250=250 ug/cm2, using a density of 1).

The NOEC of 5% in the HRIPT results in 2550 ug/cm2 (based on an application volume of 200 ul and application area at 3.63 cm2 x 0.926 density).

The summary of the LLNA is presented below and thereafter the HRIPT test.

LLNA test:

The skin sensitisation potential of the substance has been tested according to OECD TG 429: Local Lymph Node Assay" method (draft November 2000), non-GLP. At 0.1, 1, 10 and 100% the substance showed SI values of 0.9, 0.9, 10.4 and 17.5, respectively. Reliable negative and positive controls were included.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. These results show that the test substance could elicit a SI ≥ 3.

An EC3 has been derived resulted in an EC3 of 3%. A NOEC of 1% is derived.

Based on the results, the substance was considered to be a sensitiser and should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

HRIPT test:

In a repeated insult patch test, approximately 0.2 mL of 5% in Alcohol SD39C:DEP (75:25) was applied directly to an occluded patch, which was placed to the back of each subject between the scapulae and waist, adjacent to the spinal mid-line. This procedure was repeated every Monday, Wednesday and Friday until 9 applications of the test substance had been made. The patches remained in place for 24 hours and application sites were scored for dermal responses just prior to the next patch application.

Approximately 2 weeks after application of the last induction patch, a challenge patch was applied to a previously unpatched (virgin) test site. Twenty-four hours later, the patch was removed and sites were scored at 24, 48, and 72 hours after application of the challenge patch.

During the Challenge phase, bare perceptible (+) responses were observed on three (3/105) test panellists at the 24 -hour evaluation. By the 48 -hour evaluation, no response was noted. Due to the low severity and transient nature of these responses, they are not considered to be evidence of sensitisation. The NOAEC from this study is therefore 5%.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The substance is not a skin sensitiser because there are no human data that indicate respiratory skin sensitisation. In addition the potential for respiratory sensitization of the substance is assessed using the integrated evaluation strategy for respiratory sensitization data in the ECHA guidance (R7A, Fig. 7.3-2).

1)           The substance is a weak skin sensitizer;

2)           The substance does not belong to the di-isocyanates;

3)           The substance has not structural alerts or is structurally related to chemicals causing respiratory sensitization as presented in Table R.7.3-1 in the ECHA guidance of 2008 or those provided in the following document: http://ec.europa.eu/health/scientific_committees/docs/annex6_respiratory.pdf

Using this ITS in the ECHA guidance it can be concluded that Galbascone is not a respiratory sensitiser.

Justification for classification or non-classification

The substance was considered to be a sensitiser and should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction according to Regulation (EC) No. 1272/2008.