4,8-dicyclohexyl-6-hydroxy-2,10-dimethyl-12H-dibenzo[d,g][1,3,2]dioxaphosphocin

Endpoint summary

Description of key information

Skin sensitization: Local Lymph Node Assay, mice (CBA/CaOlaHsd), female (OECD guideline 429): not sensitizing

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:

2015-06-24 - 2015-07-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well-documented GLP OECD 429 Guideline study without relevant deviations on the registered substance itself.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines for Testing of Chemicals, Updated Guideline 429: Skin Sensitisation: Local Lymph Node Assay (adopted 22 July 2010).

Deviations:

yes

Remarks:

The relative humidity in the animal room was between approximately 45 - 100% instead of 45 – 65% and the temperature was between 20 and 26°C instead of 20 and 24°C for several hours, which does not affect the validity of the study.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No. 440/2008, B.42: “Skin Sensitisation: Local Lymph Node Assay”, dated 06 July 2012

Deviations:

yes

Remarks:

The relative humidity in the animal room was between approximately 45 - 100% instead of 45 – 65% and the temperature was between 20 and 26°C instead of 20 and 24°C for several hours, which does not affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CBA/CaOlaHsd mice from Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 17.8 - 21.0 g
- Housing: As group in Makrolon Type II (pre-test) / III (main study) cages, with wire mesh top and granulated soft wood bedding
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C (except for several hours, see deviations)
- Humidity (%): relative humidity approx. 45-65% (except for several hours, see deviations)
- Photoperiod (hrs dark / hrs light): 12 / 12, artificial light 6.00 a.m. - 6.00 p.m.

Vehicle:

other: tetrahydrofuran

Concentration:

1, 2.5, and 5% (w/w)
The test item was placed into an appropriate container on a tared balance and THF was added. Grinding of the test item in a mortar and warming to 37°C was used to formulate the test item.
The different test item concentrations were prepared serially.

No. of animals per dose:

2 females for each pre-test
4 females (nulliparous and non-pregnant) per dose group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 25% solution in THF, as the test item could not be formulated in any other common vehicle (tested in: acetone:olive oil 4+1 (v/v), Dimethylformamide, Methyl ethyl ketone, propylene glycol, DMSO, and cotton seed oil). Grinding of the test item in a mortar and warming to 37°C was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
- Irritation/Lymph node proliferation response: 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 10 and 25% 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.
At the tested concentrations the animals did not show any signs of systemic toxicity. From day 2 to 5, the animals treated showed a slight erythema of the ear skin (Score 1) as well as on the scalp. On day 6, upon preparation, eschar formation was visible underneath the test item residues on the ears of both animals (Score 4) and moreover the animals showed visible ear swelling. Additionally, the threshold, recommended by OECD 429, for ear thickness and ear weight was exceeded for both animals. Test item rests were observed on the ears of both animals throughout the study period. It was assumed that the observed changes were at least partially attributable to the stickiness of the test item formulations and common scratching of the animals in order to clean themselves from the adherent test item.
Therefore, a second pre-test was performed using test item concentrations of 2.5 and 5%. From day 2 to 6, the animals treated showed a slight erythema of the ear skin (Score 1) as well as on the scalp. A slight, but not excessive increase in ear thickness was observed.
Thus, the test item in the main study was assayed at 1, 2.5, and 5% (w/w). The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: ³HTdR integration
- Criteria used to consider a positive response: A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• 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:
Topical application
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 1, 2.5, and 5% (w/w) in THF. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 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).
Administration of ³H-methyl-thymidine
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 19.7 μCi of ³H-methyl thymidine (equivalent to 78.7 μCi/mL ³HTdR) were injected into each test and control mouse via the tail vein.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation).
The Dean-Dixon-Test and the Grubb’s test were used for identification of possible outliers (performed with validated program R Script Outlier.Rnw). No outlier was detected.

Positive control results:

Results of the GLP Positive Control
Experiment performed in April 2015 (Harlan study number 1690900). Positive control substance: α-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4+1, v/v)
See "Any other information on results"

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: 1281.5 (0% test item) 1825.3 (1% test item) 2674.3 (2.5% test item) 3305.1 (5% test item)

Parameter:

SI

Value:

1.42

Test group / Remarks:

1% test item

Parameter:

SI

Value:

2.09

Test group / Remarks:

2.5% test item

Key result

Parameter:

SI

Value:

2.58

Test group / Remarks:

5% test item

Remarks on result:

other:

Remarks:

highest dose tested, limited by irritating properties; test item is not sensitizing

Interpretation of results:

not sensitising

Conclusions:

The study was conducted under GLP according to OECD guideline 429 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the skin sensitization potential of CAS 73912-21-7. Under the experimental conditions reported, the test item did not induce a stimulation index over 3 in test concentrations limited by irritating effects. Therefore, CAS 73912-21-7 is considered not to be a skin sensitiser under the test conditions of this study.

Executive summary:

In a dermal sensitization study (OECD 429) with CAS 73912-21-7 in THF, 9-10 weeks old female CBA/CaOlaHsd mice were tested in a local lymph node assay. α-Hexylcinnamaldehyde served as positive control.

For this purpose a local lymph node assay was performed using test item concentrations of 1, 2.5, and 5% (w/w). 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.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. From day 2 to 4, the animals treated with a test item concentration of 5% showed an erythema of the ear skin (Score 1). Animals treated with 1 and 2.5% test item concentration did not show any signs of local skin irritation.

In this study Stimulation Indices (S.I.) of 1.42, 2.09, and 2.58 were determined with the test item at concentrations of 1, 2.5, and 5% (w/w) in THF, respectively. A dose response was observed.

In this study, CAS 73912-21-7 is not a dermal sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

There is a study available which was conducted under GLP according to OECD guideline 429 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation. Positive and negative controls gave the appropriate response. Hence, the results can be considered as reliable to assess the skin irritating potential of CAS 73912-21-7. Under the experimental conditions reported, CAS 73912-21-7 is considered not to be a skin sensitiser. There is no indicating given that the available result is not relevant for human risk assessment, the study is of high quality, no data gaps were identified. Hence, no additional testing is required and during risk assessment the substance can be regarded as not sensitizing.

Short description of key information:
Skin sensitization: Local Lymph Node Assay, mice (CBA/CaOlaHsd), female (OECD guideline 429): not sensitizing

Justification for selection of skin sensitisation endpoint:
only study available

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin Sensitisation:

In the available local lymph node assay, Stimulation Indices of 1.42, 2.09, and 2.58 were determined with the test item at concentrations of 1, 2.5, and 5% (w/w) in THF. A dose response was observed. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. As the determined S.I. is below 3, the substance does not need to be classified as skin sensitizer.