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Description of key information

In a mouse LLNA (BrdU-ELISA), conducted according to OECD guideline 442B, and to GLP, 'Karstedt concentrate' did not meet the criteria to be considered as a skin sensitiser (Haferkorn, 2016b).

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:
21 April - 24 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Species:
mouse
Strain:
CBA:JN
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier Labs, CS 4105 Le Genest, Saint Isle, Saint Berthevin, Cedex 53941, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at main study initiation: 11 weeks
- Weight at study initiation: 18-24 g
- Housing: Before application the animals were housed in groups of 5 animals in MAKROLON cages (type III) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 15 cm. After application the animals were housed singly in order to prevent them licking off the test item from the ears of the other animals. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material.
- Diet: Commercial diet ssniff® R/M-H V1534 (sourced from ssniff Spezialdiäten GmbH, 59494 Soest, Germany) was offered ad libitum. Food residue was removed.
- Water: Tap water was offered ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3 (maximum range)
- Humidity (%): 55 ± 15 (maximum range)
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 19 May 2016 To: 24 May 2016
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Pre-screening test: 0.5, 1. 2.5, 5. 10, 25, 50 or 100% (w/w)
Main study: 0, 10, 25 or 50% (w/w)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN (RANGE-FINDING) TESTS:
- Compound solubility: In a preliminary solubility assessment, the following vehicles were assessed: acetone/olive oil (4:1 v/v), N,N-dimethylformamide, Methyl ethyl ketone, Dimethyl sulfoxide and propylene glycol. Acetone/olive oil (4:1 v/v) gave homogenous solutions suitable for application of the test item and was used as the vehicle in the main study.
- Systemic toxicity and Irritation: All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination (Day 6). Both ears of each mouse were observed for erythema and scored on a 0-4 scale (0: no erythema; 1: very slight erythema (barely perceptible); 2: well-defined erythema; 3: moderate to severe erythema; 4: severe erythema (beet redness) to eschar formation preventing grading of erythema).
- Ear thickness measurements: Ear thickness measurements were taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose), and
Day 6. Additionally, on Day 6, ear thickness was determined by ear punch weight determinations, which was performed after the animals were humanely sacrificed.
Excessive local irritation would be indicated by an erythema score ≥3 and/or ear thickness of ≥25% on any day of measurement. The highest dose selected for the main LLNA: BrdU-ELISA study was selected as the next lower dose in the pre-screen concentration series that did not induce systemic toxicity and/or excessive local skin irritation.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay BrdU-ELISA
- Criteria used to consider a positive response: Results of each treatment group are expressed as the mean SI. The SI is derived by dividing the mean BrdU labelling index/mouse within each test item group and the positive control group by the mean BrdU labelling index for the solvent/vehicle control group. The average SI for the vehicle control is then one. The BrdU labelling index is defined as:
BrdU labelling index = (ABSem – ABS blankem) – (ABSref – ABS blankref)
Where: em = emission wavelength; and ref = reference wavelength
The decision process regards a result as positive when SI ≥1.6. However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses would also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9) is declared positive.

For a borderline positive response between an SI of 1.6 and 1.9, it may be necessary to consider additional information such as dose-response relationship, evidence of systemic toxicity or excessive irritation, and where appropriate, statistical significance together with SI values to confirm that such results are positives. Consideration would be also given to various properties of the test item, including whether it has a structural relationship to known skin sensitisers, whether it causes excessive skin irritation in the mouse, and the nature of the dose-response observation.
In addition, the average ear weights per group and the average ear thickness per group were compared to the vehicle control group as an indication for possible irritating properties.

TREATMENT PREPARATION AND ADMINISTRATION: groups of mice were given topical applications to the dorsum of the ear of 25 μl of formulation on three consecutive days (1,2 and 3). On day 5, animals were injected intraperitoneally with 0.5 mL (5 mg/mouse) of a 10 mg/mL solution of 5-bromo-2-deoxyuridine (BrdU) and killed 24 hours later. The draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline (PBS) for each animal.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Assessment of dose-response trends was done by linear regression, and Dunnett's test for pair-wise comparisons.
Positive control results:
Treatment with the positive control item caused the expected increases in the BrdU labelling index (see Table 1). Therefore, the study can be regarded as valid.
Key result
Parameter:
SI
Value:
< 1.6
Test group / Remarks:
10, 25 and 50%
Remarks on result:
other: Treatment with the test item at concentrations of 10%, 25% or 50% revealed a dose-related increase in the BrdU labelling index (SI values of 1.236, 1.503 and 1.588, respecitvely), but none of the SI values exceeded the threshold of 1.6.

Table 1: Stimulation indices (SI)

Parameter

Negative control

10% TM

25% TM

50% TM

Positive control

BrdU labelling index (SI)

1.000

1.236

1.503*

1.588*

1.794*

Ear punch weight (SI)

1.000

1.006

1.188

1.263

1.363*

Ear thickness increase (%, TD3)

1.0

1.4

0.0

7.3

9.6

Ear thickness increase (%, TD6)

1.9

3.7

3.3

15.7

13.4

  *Significantly increased compared to negative control at p ≤ 0.01

TM - Test material (Karstedt concentrate)

TD - Test Day

Treatment with the test item at concentrations of 10%, 25% or 50% revealed a dose-related increase in the BrdU labelling index, but not exceeding the SI threshold value of 1.6. Hence, the test item is not considered a skin sensitiser.

No signs of erythema were observed at any concentration. According to the study investigators, the "dose-related increase of the ear weight and the increase of ear thickness at the concentration of 50% may be caused by irritating properties of the test item at higher concentrations".

No mortality, clinical signs or effects on body weight were observed during the study.

Interpretation of results:
GHS criteria not met
Conclusions:
In a mouse LLNA (BrdU-ELISA) conducted according to OECD guideline 442B, and to GLP, Karstedt concentrate did not meet the critieria to be considered as a skin sensitiser.
Executive summary:

The skin sensitising potential of 'Karstedt concentrate' has been assessed in a GLP mouse local lymph node assay (LLNA): BrdU-ELISA, conducted according to OECD Test Guideline 442B. Following a preliminary range-finding study to assess irritancy, female mice CBA/JN mice (5/group) were treated topically with 0, 10, 25 or 50% Karstedt concentrate (in acetone/olive oil 4:1) on three consecutive days (1, 2 and 3). On day 6, cell proliferation in the local lymph nodes was measured by incorporation of injected 5-bromo-2-deoxyuridine (BrdU) using ELISA.

No mortality or clinical signs of toxicity were observed. No signs of erythema were observed at any test concentration (10-50%). At the highest tested concentration (50%), increases in ear weight and ear thickness were observed, which the study investigators suggest may be caused by local irritation (at higher concentrations). The BrdU labelling index resulted in stimulation index (SI) values of 1.236, 1.503 and 1.588 at test material concentrations of 10, 25 and 50% w/w, respectively. As such, a dose-related increase in the BrdU labelling index (SI) was observed. However, none of the corresponding SI values exceeded the threshold of 1.6. Positive and vehicle controls performed as expected.

Therefore, under the conditions of this study, Karstedt concentrate does not require classification for skin sensitisation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitising potential of 'Karstedt concentrate' has been assessed in a mouse local lymph node assay (LLNA): BrdU-ELISA, conducted according to OECD Test Guideline 442B and to GLP. Following a preliminary range-finding study to assess irritancy, female mice CBA/JN mice (5/group) were treated topically with 0, 10, 25 or 50% Karstedt concentrate (in acetone/olive oil 4:1) on three consecutive days (1, 2 and 3). On day 6, cell proliferation in the local lymph nodes was measured by incorporation of injected 5-bromo-2-deoxyuridine (BrdU) using ELISA.

 

No mortality or clinical signs of toxicity were observed. No signs of erythema were observed at any test concentration (10-50%). At the highest tested concentration (50%), increases in ear weight and ear thickness were observed, which the study investigators suggest may be caused by local irritation (at higher concentrations). Observed stimulation index values were 1.236, 1.503 and 1.588 at test material concentrations of 10, 25 and 50% w/w, respectively. A dose-related increase in the BrdU labelling index was observed. However, none of the corresponding SI values exceeded the threshold of 1.6. Positive and vehicle controls performed as expected. Therefore, under the conditions of this study, 'Karstedt concentrate' does not require classification for skin sensitisation (Haferkorn, 2016b).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

No respiratory tract sensitisation data were identified. A new study was not conducted as it is not a REACH Standard Information Requirement.

Justification for classification or non-classification

Based on the results of the available and reliable murine LLNA assay, ‘Karstedt concentrate’ does not warrant classification for skin sensitisation, according to EU CLP criteria (EC 1272/2008).

No known information exists which would necessitate classification for respiratory sensitisation.