Reaction mass of (4R)-isopropenyl-1-methylcyclohexene and (4S)-isopropenyl-1-methylcyclohexene and 1-isopropyl-4-methylcyclohexa-1,3-diene and 1-isopropyl-4-methylcyclohexa-1,4-diene and 4-isopropylidene-1-methylcyclohexene

Endpoint summary

Description of key information

In a local lymph node assay (LLNA) performed according to OECD Guideline 429 and in compliance with GLP, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was found to be a skin sensitiser.

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:

13 March to 10 April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

10 January 2013

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Preliminary test - Approximately 10 weeks; Main test - Approximately 8 weeks
- Weight at study initiation: Preliminary test - 22.7 g (22.3-23.1 g); Main test - 19.9 g (18.3-22.0 g)
- Housing: Animals were housed by group of two (preliminary test) or four (main test) in polycarbonate cages (Tecniplast 1145T, 435 cm2) containing autoclaved sawdust (SICSA, Alfortville, France). In the main test, on Day 6 before the 3H-TdR injections, the animals were housed in disposable crystal polystyrene cages (22.0 cm x 8.5 cm x 8.0 cm).
- Diet: SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Tap water (filtered using a 0.22 µm filter), ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20%
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 h dark / 12 h light

Vehicle:

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

Concentration:

Preliminary test: 10, 25, 50 and 100 %
Main test: 2.5, 5, 10, 25 and 50 %

No. of animals per dose:

Preliminary test: One concentration per ear (10 and 25 % in left and right ear of 2 animals, respectively; 50 and 100 % in left and right ear of 2 animals, respectively)
Main test: 4 females/dose

Details on study design:

PRELIMINARY TEST:
- Compound solubility: A solution was obtained at the concentration of 50 % in AOO.
- As the test item is a liquid that could be sampled using a pipette, the maximum achievable concentration was 100 %.
- A preliminary test was performed in four animals to assess the irritant potential of the test item (through ear thickness measurement). On Days 1, 2 and 3, a dose-volume of 25 μL of the appropriate dose formulation was applied to the dorsal surface of both ears (one concentration per ear), using an adjustable pipette fitted with a plastic tip.
- Irritation: Dryness of ear skin was noted in both females treated at 50 and 100 % on day 6. No local reactions were observed at the concentrations of 25 and 10 %. A slight increase in ear thickness (< 25 %) was recorded in one female at the concentration of 25 % and in one other female at the concentration of 50 %. An excessive local skin irritation (increase in ear thickness > 25 %) was noted in both females treated at 100 %, showing the irritant potential of the test item at this concentration. The highest concentration retained for the main test was therefore 50 %.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT:
- Animals were allocated to the groups using a manual randomization procedure.
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item is considered as a skin sensitizer when the SI for a dose group is ≥ 3 together with consideration of a dose-response relationship. Other relevant criteria such as radioactivity levels and ear thickness are also taken into account to evaluate the data.

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of control and test item were applied to the dorsal surface of both ears on Days 1, 2 and 3. On Day 6, 250 µL of 0.9 % NaCl containing 20 μCi of 3H-TdR (specific activity of 20 Ci/mmol) was injected into the tail vein of each experimental mouse. Five hours later, all mice were sacrificed by an intraperitoneal injection of pentobarbital sodium followed by a cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group. A single cell suspension of auricular lymph node cell (ALNC) was prepared by mechanical disaggregation in Petri dishes using the plunger of a syringe. Cell suspensions were washed with 0.9 % NaCl and precipitated with 5 % (w/v) trichloroacetic acid (TCA) at 4 °C. 3 mL of Ultima GoldxR scintillation fluid (Packard) was added in order to measure incorporation of 3H-TdR using β-scintillation counting.
The results were expressed as disintegrations per minute (dpm) per group and as dpm/node.

Stimulation Index (SI) were calculated according to the following formula:
SI = dpm per node of the treated group / dpm per node of the vehicle control group

Positive control substance(s):

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

Statistics:

No data

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group (SI = 12.35). The experiment was therefore considered valid.

Key result

Parameter:

SI

Remarks on result:

other: Stimulation index for 2.5, 5, 10, 25 and 50 % were 1.02, 0.87, 4.90, 8.97 and 20.10, respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: - DPM per group for vehicle, 2.5, 5, 10, 25 and 50 % were 1760, 1787, 1527, 8617, 15792 and 35373, respectively. - DPM per node for vehicle, 2.5, 5, 10, 25 and 50 % were 220.00, 223.38, 190.88, 1077.13, 1974.00 and 4421.63, respectively.

Table 7.4.1/1: Skin sensitisation – results

 

Treatment and concentrations	Number of nodes per group	dpm per group	dpm per node	Stimulation index (SI)	Increase in ear thickness (% between day 1 and day 6)	Irritation level	EC3 value (%)
Vehicle	8	1760	220.00	-	0.00	-	-
Test item 2.5 %	8	1787	223.38	1.02	2.00	I	7.64
Test item 5 %	8	1527	190.88	0.87	0.00	I
Test item 10 %	8	8617	1077.13	4.90	0.00	I
Test item 25 %	8	15792	1974.00	8.97	1.98	I
Test item 50 %	8	35373	4421.63	20.10	11.11	II
HCA 25 %	8	21738	2717.25	12.35	-	-	-

 

dpm = disintegrations per minute

HCA = α-hexylcinnamaldehyde

I = non-irritant (increase in ear thickness < 10 %)

II = slightly irritant (increase in ear thickness 10 to 25 %)

 

EC3 value = theoretical concentration resulting in a SI value of 3

 

Mortality and clinical signs: No unscheduled deaths or clinical signs were observed in any animals.

Body weight: The body weight change of test item-treated animals was similar to that of control animals.

Ear thickness measurements and local reactions:Dryness of ear skin was noted in all females treated at 50 % on day 6. No local reactions were observed in animals treated at lower dose-levels. A slight increase in mean ear thickness of 11 % was observed in females treated at 50 %.

 

Proliferation assay:

A dose-related increase in the SI was recorded at concentrations ≥ 10 %, and the threshold positive value of 3 was exceeded at the concentration of 10 % (SI = 4.90). In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

The EC3 value is equal to 7.64 %.

Interpretation of results:

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

Conclusions:

The test item is classified in category 1 and sub-category 1B according to CLP Regulation (EC) No. 1272/2008.

Executive summary:

In a local lymph node assay performed according to OECD Guideline 429 and in compliance with GLP, groups of CBA/J mice (4 females/dose) were topically applied with 25 µL of test item REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE at concentrations of 2.5, 5, 10, 25 and 50% to the dorsal surface of both ears for three consecutive days. Vehicle control group of four females received the vehicle (acetone/olive oil (4/1; v/v)). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (Day 6). The results were expressed as disintegrations per minute (dpm) per group; dpm/node and the obtained values were used to calculate Stimulation Indices (SI). Animals were observed for mortality, clinical signs and body weight during the study. The test concentrations for the main study were determined from a preliminary study (10, 25, 50 and 100 %; one concentration per ear in two animals).

In the main test, no unscheduled deaths and no clinical signs were observed during the observation period. Body weight of animals was unaffected by the test item treatment. Dryness of ear skin was noted in all females treated at 50% on day 6. No local reactions were observed in animals treated at lower dose-levels. A slight increase in mean ear thickness of 11% was observed in females treated at 50%.

 

The observed SI values were 1.02, 0.87, 4.90, 8.97 and 20.10 at 2.5, 5, 10, 25 and 50%, respectively. A dose-related increase in the SI was recorded at concentrations ≥ 10%, and the threshold positive value of 3 was exceeded at the concentration of 10% (SI = 12.35). In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

The EC3 value is equal to 7.64%.

 

Therefore, the test item is classified in category 1 and sub-category 1B according to CLP Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In a local lymph node assay (LLNA) performed according to OECD Guideline 429 and in compliance with GLP, REACTION MASS OF DL-LIMONENE, ALPHA- GAMMA- TERPINENES, TERPINOLENE was found to be a skin sensitiser.

The obtained SI values were 1.02, 0.87, 4.90, 8.97 and 20.10 at 2.5, 5, 10, 25 and 50 %, respectively.

A dose-related increase in the SI was recorded at concentrations ≥ 10%, and the threshold positive value of 3 was exceeded at the concentration of 10% (SI = 12.35). In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value is equal to 7.64 %. Therefore, the test item is classified in category 1 and sub-category 1B according to CLP Regulation (EC) No. 1272/2008.