Reaction mass of 2-(cis-4-methylcyclohexyl)propan-2-ol and 2-(trans-4-methylcyclohexyl)propan-2-ol and cis-1-methyl-4-(propan-2-yl)cyclohexanol and trans-1-methyl-4-(propan-2-yl)cyclohexanol

Endpoint summary

Description of key information

In a GLP study conducted in compliance with OECD Guideline 429 with the registered substance, an EC3 value of 95% was obtained.

In a confirmatory study conducted in compliance with OECD Guideline 4442B with the registered substance, an EC1.6 value of 75.53% was obtained.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 February - 03 March 2014

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:

other: 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 - 21.3 g (20.4-22.5 g); Main test - 19.7 g (18.0-22.1 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: 5, 10, 25, 50 and 100 %

No. of animals per dose:

Preliminary test: One concentration per ear (10 and 25 % in left ear of 1 female each; 50 and 100 % in right ear of 1 female each)
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 was 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 skin was noted on Day 6 in right ear of all group one females (test item at 100 %). At the concentration of 100 %, an increase in ear thickness of 16 % was noted. This slight increase in ear thickness was comprised between 10 and 25 %. The test item was then considered as slightly irritant at this concentration. However, as the acceptance limit of 25 % was not reached, this concentration was not rejected for testing on the main test. Increases in ear thickness at the lowest tested concentrations (i.e. 10, 25 and 50 %) were all below 10 %. The highest concentration retained for the main test was therefore 100 %.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT:
- Animals were allocated to the groups using a computerized 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 to 21 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 = 4.69). The experiment was therefore considered valid.

Parameter:

SI

Remarks on result:

other: Stimulation index for 5, 10, 25, 50 and 100 % were 1.29, 0.72, 0.66, 2.27 and 3.09, respectively.

Key result

Parameter:

EC3

Remarks:

%

Value:

95

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	2173	271.63	-	-4.85	-	-
Test item 5 %	8	2798	349.75	1.29	-5.00	I	95
Test item 10 %	8	1572	196.50	0.72	-5.81	I
Test item 25 %	8	1425	178.13	0.66	-7.85	I
Test item 50 %	8	4942	617.75	2.27	-4.00	I
Test item 100 %	8	6708	838.50	3.09	-1.92	I
HCA 25 %	8	10193	1274.13	4.69	-	-	-

 

dpm = disintegrations per minute

HCA = α-hexylcinnamaldehyde

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

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: No local reactions were observed in any animals.

In all test item-treated groups, the increase in ear thickness was below 10 %. The test item was therefore considered as non-irritant. As the acceptance limit of 25 % was not reached at any concentrations, no bias in the results of proliferation measurements linked to an excessive irritant potential was considered.

 

Proliferation assay:

No significant lymphoproliferation was noted at concentrations 5, 10 and 25 %. A slight increase of the stimulation indices was noted from the concentration 50 % (SI = 2.27), reaching the threshold positive value at 100 % (SI = 3.09 %).

In the absence of local irritation, the slight significant lymphoproliferative response observed may be attributed to delayed contact hypersensitivity.

The EC3 value was equal to 95%.

Taking into account that the significant lymphoproliferation was obtained for the highest concentration, with a stimulation index equal to the positive threshold, further investigations could be considered to confirm the very weak response noted in this study.

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) N° 1272/2008.
However, the stimulation index threshold positive value of 3 was reached only at the highest tested concentration of 100% and did not clearly exceed this threshold value. Therefore, a false positive result cannot be excluded and further experiments could be considered to confirm or to contradict the obtained results.

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 dihydroterpineol multiconstituent at concentrations of 5, 10, 25, 50 and 100% 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 (one concentration per ear (10 and 25% in left ear of 1 female each; 50 and 100% in right ear of 1 female each)).

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. No local reactions were observed in any animals. In all test item treated groups, the increase in ear thickness was below 10 %. The test item was therefore considered as non-irritant. As the acceptance limit of 25% was not reached at any concentrations, no bias in the results of proliferation measurements linked to an excessive irritant potential was considered.

 

The SI of the positive control was > 3 (4.69%); this experiment was therefore considered valid.

 

The observed SI values were 1.29, 0.72, 0.66, 2.27 and 3.09 at 5, 10, 25, 50 and 100%, respectively. The threshold positive value of 3 was reached at the concentration 100%. In the absence of local irritation, the significant lymphoproliferative response observed was attributed to delayed contact hypersensitivity. The EC3 value was equal to 95%.

 

Therefore, the test item is classified in category 1 and sub-category 1B according to CLP Regulation (EC) N° 1272/2008.

However, the stimulation index threshold positive value of 3 was reached only at the highest tested concentration of 100% and did not clearly exceed this threshold value. Therefore, a false positive result cannot be excluded and further experiments could be considered to confirm or to contradict the obtained results.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05-25 August 2015

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 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

23 October 2015

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J (CBA/JRj)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier Labs, Le Genest-Saint-Isle, France.
- Age at study initiation: 8 weeks
- Weight at study initiation: 19.7-23.5 g
- Housing: Animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood wood flakes.
- Diet: Foodstuff (A04, SAFE), ad libitum
- Water: Drinking water (tap water from public distribution system), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 10 changes/h
- Photoperiod: 12 h dark / 12 h light

Vehicle:

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

Concentration:

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

No. of animals per dose:

Preliminary test: Two animals
Main test: 4 females/dose

Details on study design:

PRELIMINARY TEST:
- The mice were treated by daily application of 25 μL of the test item undiluted (100 %) or diluted at 50 % in Acetone/olive oil (4:1, v/v) to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed daily from day 1. Any signs of toxicity or excessive local irritation noted during this period were recorded. Ear thickness was recorded on Days 1, 3 and 6.
- No cutaneous reactions were noted at the concentration of 100 %. Therefore this concentration was chosen as the highest concentration for the main study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT:
- Animals were randomly allocated to the groups.
- Name of test method: Local Lymph Node Assay (LLNA:BrdU)
- Criteria used to consider a positive response: Test item should be classified if at least one concentration of the test item results in a SI≥ 3, as sensitiser. Any test item failing to produce a SI> 1.6 was classified as a "non-sensitiser".

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 5, 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route. On day 6 (end of the test), the animals were were sacrificed by pentobarbital sodium and the auricular lymph nodes were excised. Then, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte with ELISA kit.

Positive control substance(s):

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

Statistics:

No data

Positive control results:

The Stimulation Index (SI) for the 25 % HCA treated group was 1.83. Therefore, the positive control is classified as sensitiser, as expected, and the validity of the study was confirmed.

Parameter:

SI

Remarks on result:

other: Stimulation Index was 0.86, 0.88, 1.17, 1.36 and 1.83 for the treated groups at 5, 10, 25, 50 and 100 %, respectively.

Key result

Parameter:

other: EC1.6

Remarks:

%

Value:

75.53

Clinical observations and mortality

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.

Bodyweights

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Local irritation

Dryness of the skin was noted on Day 6 in all animals treated with undiluted test item.

No significant increase in ear thickness and in ear weight was noted in animals treated at 5, 10, 25, 50 and 100 %. Therefore, the test item has to be considered as not excessively irritant at these concentrations.

 

Table 7.4.1/1: BrdU index & Stimulation index per group and calculation of EC_1.6

Groups	Test item	Brdu-index (Mean*)	Stimulation Index (SI) (Mean ± SD)	Result	EC1.6 value
1	AOO	0.677	NA	NA	NA
2	5	0.584	0.86 ± 0.09	Negative	75.53 %
3	10	0.598	0.88 ± 0.15	Negative
4	25	0.794	1.17 ± 0.23	Negative
5	50	0.923	1.36 ± 0.09	Negative
6	100	1.240	1.83 ± 0.15	Positive
7	HCA 25 %	1.238	1.83 ± 0.23	Positive	NA

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 in accordance with CLP Regulation (EC) N° 1272/2008.

Executive summary:

In a local lymph node assay (LLNA:BrdU) performed according to OECD Guideline 442B and in compliance with GLP, groups of CBA/J mice (4 females/dose) were topically applied with 25 µL of test item dihydroterpineol multiconstituent at concentrations of 5, 10, 25, 50 and 100% 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)). On Day 5, 0.5 mL of BrdU solution (10 mg/mL) was injected by intra-peritoneal route. On Day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte with ELISA kit and Stimulation Indices (SI) were calculated. 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 (50 and 100 %).

No mortality and no signs of systemic toxicity were noted in the treated and control animals during the test. Dryness of the skin was noted on Day 6 in all animals treated with undiluted test item. No significant increase in ear thickness and in ear weight was noted in animals treated at 5, 10, 25, 50 and 100%. Therefore, the test item has to be considered as not excessively irritant at these concentrations.

Stimulation Index was 0.86, 0.88, 1.17, 1.36 and 1.83 for the treated groups at 5, 10, 25, 50 and 100 %, respectively. The EC1.6 was determined to be 75.53%.

The result of the study is positive due to the fact that the SI is higher than 1.6 in the treated group at 100% and that the SI value is the same as the positive control. Furthermore a dose-response relationship was noted.

The Stimulation Index (SI) for the 25% HCA treated group was 1.83. Therefore, the positive control is classified as sensitiser, as expected, and the validity of the study was confirmed.

Therefore, the test item is classified in category 1 and sub-category 1B in accordance with CLP Regulation (EC) N° 1272 /2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

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 dihydroterpineol multiconstituent at concentrations of 5, 10, 25, 50 and 100% 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 (one concentration per ear (10 and 25 % in left ear of 1 female each; 50 and 100 % in right ear of 1 female each)).

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. No local reactions were observed in any animals. In all test item-treated groups, the increase in ear thickness was below 10%. The test item was therefore considered as non-irritant. As the acceptance limit of 25% was not reached at any concentrations, no bias in the results of proliferation measurements linked to an excessive irritant potential was considered.

The SI of the positive control was >3 (4.69 %); this experiment was therefore considered valid.

The observed SI values were 1.29, 0.72, 0.66, 2.27 and 3.09 at 5, 10, 25, 50 and 100 %, respectively. The threshold positive value of 3 was reached at the concentration 100%. In the absence of local irritation, the significant lymphoproliferative response observed was attributed to delayed contact hypersensitivity. The EC3 value was equal to 95%.

However, the stimulation index threshold of 3 was reached only at the highest tested concentration of 100% and did not clearly exceed this threshold value. As a false positive result could not be excluded, another study was conducted in a different laboratory and according to a different but closely related methodology, with individual assessment in order to better characterise the potential dose-response.

In a local lymph node assay (LLNA:BrdU) performed according to OECD Guideline 442B and in compliance with GLP, groups of CBA/J mice (4 females/dose) were topically applied with 25 µL of test item dihydroterpineol multiconstituent at concentrations of 5, 10, 25, 50 and 100% 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)). On Day 5, 0.5 mL of BrdU solution (10 mg/mL) was injected by intra-peritoneal route. On Day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte with ELISA kit andStimulation Indices (SI) were calculated. 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 (50 and 100%).

No mortality and no signs of systemic toxicity were noted in the treated and control animals during the test. Dryness of the skin was noted on Day 6 in all animals treated with undiluted test item. No significant increase in ear thickness and in ear weight was noted in animals treated at 5, 10, 25, 50 and 100%. Therefore, the test item has to be considered as not excessively irritant at these concentrations.

Stimulation Index was 0.86, 0.88, 1.17, 1.36 and 1.83 for the treated groups at 5, 10, 25, 50 and 100%, respectively. The EC1.6 was determined: 75.53%.

The result of the study is positive due to the fact that the SI is higher than 1.6 in the treated group at 100% and that the SI value is the same as the positive control. Furthermore a dose-response relationship was noted.

The Stimulation Index (SI) for the 25% HCA treated group was 1.83. Therefore, the positive control is classified as sensitiser, as expected, and the validity of the study was confirmed.

Therefore, the test item is classified in category 1 and sub-category 1B in accordance with CLP Regulation (EC) N° 1272 /2008.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In two in vivo studies conducted according to OECD guideline 429 and OECD guideline 442B with the registered substance, the EC3 and EC1.6 were much higher than 2% w/w, therefore the registered substance is classified in category 1 and sub-category 1B in accordance with CLP Regulation (EC) N° 1272 /2008.