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Administrative data

Description of key information

According to results from a Local Lymph Node Assay performed according to Guideline OECD 429 Nopol has to be classified in category 1 and sub-category 1B according to CLP Regulation (EC) No. 1272/2008.

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:
12 February - 06 March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline 429 without any deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
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. certificate)
Remarks:
10 January 2013
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
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 9 weeks; Main test - Approximately 8 weeks
- Weight at study initiation: Preliminary test - 20.0 g (19.7-20.1 g); Main test - 20.0 g (18.0-22.5 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: 30-70 %
- 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: No local reactions were observed in any animals. At a concentration of 50 %, a mean increase of 10.25 % in ear thickness was noted. As such, the test item was considered as slightly irritant at this concentration. For the other concentrations (10, 25 and 100 %), increases in ear thickness were all below 10 %. Since the acceptance limit of 25 % was not reached at any concentrations, the highest concentration retained for the main test was 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 = 7.09). The experiment was therefore considered valid.
Key result
Parameter:
SI
Remarks on result:
other: Stimulation index for 5, 10, 25, 50 and 100 % were 1.18, 2.19, 3.03, 5.16 and 6.21, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: - DPM per group for vehicle, 5, 10, 25, 50 and 100 % were 1284, 1513, 2807, 3891, 6631 and 7974, respectively. - DPM per node for vehicle, 5, 10, 25, 50 and 100 % were 160.50, 189.13, 350.88, 486.38, 828.88 and 996.75, 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

1284

160.50

-

-2.09

-

-

Test item

5 %

8

1513

189.13

1.18

-6.00

I

24

Test item

10 %

8

2807

350.88

2.19

-3.04

I

Test item

25 %

8

3891

486.38

3.03

-3.04

I

Test item

50 %

8

6631

828.88

5.16

5.08

I

Test item

100 %

8

7974

996.75

6.21

1.96

I

HCA

25 %

8

9104

1138.00

7.09

-

-

-

 

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:

A dose-related increase in the SI was recorded at all concentrations. At ≥ 25 %, SI values were above the cut-off value of 3. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value was equal to 24 %.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item is classified in category 1B (H317) according to CLP Regulation (EC) No. 1272/2008 and GHS.
Executive summary:

In a local lymph node assay performed according to Guideline OECD 429 and in compliance with GLP, groups of CBA/J mice (4 females/dose) were topically applied with 25 µL of the test item, Nopol, 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 (7.09 %); this experiment was therefore considered valid.

 

The observed SI values were 1.18, 2.19, 3.03, 5.16 and 6.21at 5, 10, 25, 50 and 100 %, respectively. A dose-related increase in the SI was recorded at all concentrations. At ≥ 25 %, SI values were above the cut-off value of 3. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value was equal to 24 %.

 

Therfore, the test item is classified in category 1B (H317) according to CLP Regulation (EC) No. 1272/2008 and GHS.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In a local lymph node assay performed according to Guideline OECD 429 and in compliance with GLP, groups of CBA/J mice (4 females/dose) were topically applied with 25 µL of the test item, Nopol, 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)).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Nopol has to be classified in category 1 and sub-category 1B according to CLP Regulation (EC) No. 1272/2008.