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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 April 2019 - 14 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
6-methyl-3,4-dihydro-2H-1,4-benzoxazine
EC Number:
837-106-9
Cas Number:
71472-57-6
Molecular formula:
C9H11NO
IUPAC Name:
6-methyl-3,4-dihydro-2H-1,4-benzoxazine
Test material form:
liquid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 11 weeks
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 ± 2°C, relative humidity approx. 45-65%, artificial light 6.00 a.m. - 6.00 p.m.
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Low Dose: 2.5%
Mid Dose: 5%
High Dose: 10%
No. of animals per dose:
Four
Details on study design:
PRE-SCREEN TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was 100% (undiluted test item).
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6
Both animals showed an erythema of the ear skin (Score 1 to 2) and erythema of the scalp. The animals had to be sacrificed on day 2 and 3, respectively, due to clear symptoms of toxicity such as hunched posture, piloerection, partially closed eyes, sunken flanks, ataxia, apathy, laboured breathing, and weight loss. Additionally, staining of the urine was observed (orange).
Therefore, a second pre-test was performed using test item concentrations of 10 and 25%. Both animals showed an erythema of the ear skin (Score 1 to 2) and erythema of the scalp. Clear symptoms of toxicity were observed in the animal treated with 25% of the test item which included hunched posture, partially closed eyes, laboured respiration, and decreased activity. Furthermore, a loss in body weight of >10% was observed in this animal. Therefore, this concentration was considered to be inappropriate for use in the main study. In the animal treated with 10% of the test item, only mild and transient symptoms of toxicity were observed, namely partially closed eyes 1 hour after the third application, and hunched posture on days 4-6. Only a negligible loss in body weight (3%) was noted in this animal.

MAIN STUDY
The test item in the main study was assayed at 2.5, 5, and 10%. The highest concentration tested was the highest level that could be achieved whilst avoiding clear systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

ANIMAL ASSIGNMENT AND TREATMENT
The animals were distributed into the test groups at random.
- Criteria used to consider a positive response:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and acetone/olive oil (4+1, v/v) was added (weight per weight). The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion.
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% in acetone/olive oil (4+1, v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø ~ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
All calculations were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.

Results and discussion

Positive control results:
Experiment performed in April 2019 (Envigo study number 1950400). Positive control substance: α-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4:1 v/v))
(See table 1)

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Control
Key result
Parameter:
SI
Value:
2.59
Test group / Remarks:
2.5% test item concentration
Key result
Parameter:
SI
Value:
4.48
Test group / Remarks:
5% test item concentration
Key result
Parameter:
SI
Value:
7.1
Test group / Remarks:
10% test item concentration
Key result
Parameter:
EC3
Value:
3.04
Test group / Remarks:
EC3 value calculated was 3.04 % (w/w)
Cellular proliferation data / Observations:
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

EC3 CALCULATION :
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

CLINICAL OBSERVATIONS: All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.

BODY WEIGHTS: The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment).

Any other information on results incl. tables

Table 1. Results of positive control

Test item concentration %

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

15

---

---

---

---

---

BG II

24

---

---

---

---

0

1

6866

6846.5

8

855.8

1.00

5

2

10186

10166.5

8

1270.8

1.48

10

3

15511

15491.5

8

1936.4

2.26

25

4

55457

55437.5

8

6929.7

8.10

1 = Control Group

2-4 = Test Group

a)= The mean value was taken from the figures BG I and BG II

b)= Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test substance was found to be a skin sensitiser under the test conditions of this study.
Executive summary:

In the study the test item formulated in acetone/olive oil (4+1, v/v) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments.

No cases of mortality were observed. The animals treated with the test item showed an erythema of the ear skin (Score 1 to 2) and erythema of the scalp and scaly ears. Additionally, in animals treated with the high dose of the test item only, mild clinical symptoms such as partially closed eyes, hunched posture, and decreased activity as well as staining of the urine (orange) were observed transiently on test days 2, 3, and 4.

In this study Stimulation Indices (S.I.) of 2.59, 4.48, and 7.10 were determined with the test item at concentrations of 2.5, 5, and 10% in acetone/olive oil (4+1, v/v), respectively. A clear dose response was observed.

The test substance was found to be a skin sensitiser and an EC3 value of 3.04% (w/w) was derived.