Bis(methylcyclohexyl) phthalate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2010

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

Principles of method if other than guideline:

The study procedure was extended to include additional measures of lymph node response including lymph node cell count, lymph node weight and ear weight as an indicator of skin irritation to put the data into perspective.
The basis for these additions are described in:
- Kolle et al., Performance standards and alternative assays: practical insights from skin sensitization. Regul Toxicol Pharmacol. 2013 Mar; 65(2):278-85.
- Kolle et al., Further experience with the local lymph node assay using standard radioactive and nonradioactive cell count measurements. J Appl Toxicol. 2012 Aug; 32(8):597-607.
- Basketter et al., Experience with local lymph node assay performance standards using standard radioactivity and nonradioactive cell count measurements. J Appl Toxicol. 2012 Aug;32(8):590-6.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(methylcyclohexyl) phthalate

EC Number:

248-765-1

EC Name:

Bis(methylcyclohexyl) phthalate

Cas Number:

27987-25-3

Molecular formula:

C22H30O4

IUPAC Name:

bis(1-methylcyclohexyl) phthalate

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material:KN53200656
- Expiration date of the lot/batch: 2017-11-18
- Purity test date: 2017-02-13

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient, protected from light

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, Netherlands
- Age at study initiation: 8 weeks
- Weight at study initiation: 18 - 21.2g
- Housing: single
- Diet (e.g. ad libitum): ad lib.
- Water (e.g. ad libitum): ad lib.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

10%, 50%, undilluted

No. of animals per dose:

5

Details on study design:

Dose Selection Rational:

In a pre-test, two mice per group were treated with testsubstance concentrations of 10%, 50% and the undiluted test substance on three consecutive days.
Clinical signs were recorded after each application as well as on day 5. Signs of local irritation were recorded on day 1, 2 and 5. Prior to the first application of the test item (day 0), on day 2 and before sacrifice (day 5) the ear thickness was determined by using a micrometer. Furthermore, the ears were punched after sacrifice at the apical area by using a biopsy punch (Ø 0.8 cm) and were immediately pooled per animal and weighed by using an analytical balance. Additionally, the weight of the pooled lymph nodes from both sides was determined for each animal.
No signs of systemic toxicity were observed in the pretest. At the tested concentrations, the animals did not show relevant signs of local irritation as confirmed by determination of the ear weights (compared to current vehicle values) and ear thickness measurements. Slight swelling of the ear skin was noted at all concentrations tested, during the observation period. Very slight erythema was observed in the animals, treated with the undiluted test substance from study day 1 up to study day 5. In addition, accumulation of test item with accompanying distinct erythema (head- back region) was noted after application of the undiluted test substance on study day 2 and 5.

Positive control substance(s):

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

Results and discussion

In vivo (LLNA)

Results

Parameter:

EC3

Value:

29.4

Cellular proliferation data / Observations:

SI
10%: 1.35
50%: 4.75
100%: 8.17

Lymph node cell counts
10%: 1.42
50%: 2.01
100%: 2.59

Lymph node weight
10%: 1.16
50%: 1.59
100%: 2.01

Any other information on results incl. tables

The test-substance concentrations did not cause biologically relevant increases (SI > 1.25) in ear weights demonstrating the absence of relevant ear skin irritation. However, a statistically significant increase in ear weights was noted at 50% (+5%) and 100% (+13%).

Slight swelling of the ear skin was observed in all test-substance treated animals, while severe adhesion of the test item producing distinct erythema (head/shoulder/neck region) was noticed after application of the 50% test-substance preparation and of the undiluted test substance, on study day 1 and/or 2, only.

Applicant's summary and conclusion

Interpretation of results:

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