6H-dibenz[c,e][1,2]oxaphosphorin 6-oxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of preliminary test: 04 May 2011; Start of experiment: 18 May 2011; End of experiment: 24 May 2011

Reliability:

1 (reliable without restriction)

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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:

HCA

IUPAC Name:

HCA

Constituent 2

Reference substance name:

6H-dibenz[c,e][1,2]oxaphosphorin 6-oxide

EC Number:

252-813-7

EC Name:

6H-dibenz[c,e][1,2]oxaphosphorin 6-oxide

Cas Number:

35948-25-5

Molecular formula:

C12H9O2P

IUPAC Name:

6H-dibenz[c,e][1,2]oxaphosphorin 6-oxide

Constituent 3

Reference substance name:

6H-dibenz [c,e] [1,2] oxaphosphorin 6-oxide

IUPAC Name:

6H-dibenz [c,e] [1,2] oxaphosphorin 6-oxide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): HCA
- Molecular formula (if other than submission substance): C12H9O2P
- Molecular weight (if other than submission substance): 216.18
- Physical state: White powder
- Analytical purity: >98%
- Lot/batch No.: 00423
- Expiration date of the lot/batch: April 2015
- Storage condition of test material: Room temperature (15-25°C)
- Other:
- Manufacture date: April 2010
- Safety precautions: Routine safety precautions (lab coat, gloves, goggles, face mask) for unknown materials will be applied to assure personnel
health and safety.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J Rj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source:
Elevage Janvier,
Route des Chènes Secs B.P. 4105,
53940 Le Genest-St-Isle,
France
- Age at study initiation: 9-10 weeks old (Female, nulliparous, non pregnant)
- Weight at study initiation: 21.1 – 22.7 g (The weight variation in animals involved in the study did not exceed ± 20 % of the mean weight)
- Housing:
Housing / Enrichment: Group caging / mice were provided with glass tunnel-tubes
Cage type: Type II. polypropylene/ polycarbonate
Bedding: Bedding was available to animals during the study
- Diet (e.g. ad libitum): Animals received ssniff SM R/M-Z+H "Autoclavable complete diet for rats and mice - breeding and maintenance" (Batch number: 555 5267 Expiry Date: August 2011) produced by ssniff Spezialdiaten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum.
- Water (e.g. ad libitum): Animals received tap water from the municipal supply from 500 ml bottle, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at LAB Research Ltd.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): Light - 12 hours daily, from 6.00am to 6.00pm

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

N,N-dimethylformamide

Concentration:

25, 10 and 5 (w/v)%

No. of animals per dose:

4 animals/treatment group

Details on study design:

RANGE FINDING TESTS: The Preliminary Irritation/Toxicity Test was performed in CBA/J Rj mice using two doses (test item concentrations of 25 and 10 (w/v) %) in the selected vehicle. The observations recorded in the preliminary test suggest that the 25 (w/v)% formulation is a suitable dose level for a valid LLNA. See Appendix of attached report for results of preliminary irritation/toxicity test.

MAIN STUDY:
ANIMAL ASSIGNMENT AND TREATMENT
- Identification: A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of LAB Research Ltd’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software according to the actual body weights, verifying the homogeneity and variability between the groups.

TREATMENT PREPARATION AND ADMINISTRATION:
- ADMINISTRATION OF THE TEST ITEM
- Topical application: During the assay each mouse was topically dosed on the dorsal surface of each ear with 25 µl of the appropriate formulation applied using a pipette. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Positive control substance(s):

other: α-Hexylcinnamaldehyde, ≥95%

Results and discussion

Positive control results:

Positive control 25% α-HCA in DMF:
Measured DPM/Group: 14806
Group DPM: 14764.5
No. of Nodes: 8
DPN: 1845.6
Stimulation Index Values: 16.6

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Negative control DMF: 1.0 HCA 25 (w/v)% in DMF: 2.5 HCA 10 (w/v)% in DMF: 4.4 HCA 5 (w/v)% in DMF: 4.2 Positive control 25% α-HCA in DMF: 16.6

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Measured DPM/group: Background (5 (w/v)% TCA): 41.5 Negative control DMF: 931 HCA 25 (w/v)% in DMF: 2278 HCA 10 (w/v)% in DMF: 3974 HCA 5 (w/v)% in DMF: 3804 Positive control 25 % α-HCA in DMF: 14806 Group DPM: Negative control DMF: 889.5 HCA 25 (w/v)% in DMF: 2236.5 HCA 10 (w/v)% in DMF: 3932.5 HCA 5 (w/v)% in DMF: 3762.5 Positive control 25% α-HCA in DMF: 14764.5

Any other information on results incl. tables

CLINICAL OBSERVATION: No mortality or signs of systemic toxicity were observed during the study, no local dermal effects were seen at the site of application. The results are given in Appendix 3 of attached report.

 

BODY WEIGHT MEASUREMENT: No treatment related effects were observed on animal body weights. Individual and mean body weights are given in Table 3 of attached report.

 

PROLIFERATION ASSAY: The results of the proliferation assay are summarized in Table 4 and Figure 1 of the attached report. Appearance of the lymph nodes was normal in the negative control group and in the test item treated groups. Larger than normal lymph nodes was observed in the positive control group in the 25 and 10 (w/v)% treated groups.

 

INTERPRETATION OF OBSERVATIONS: The test item was a white powder, which was dissolved in N,N-dimethylformamide.

 

Since there were no confounding effects of irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. A significant lymphoproliferative response (SI≥3) was noted for HCA at concentrations of 10 and 5 (w/v)%. Stimulation index values of the test item were 2.5, 4.4 and 4.2 at treatment concentrations of 25, 10 and 5 (w/v)%, respectively (Figure 1 of attached report).

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, under the conditions of the present assay HCA (Batch No.: 00423), tested in a suitable vehicle, was shown to have a sensitization potential (sensitizer) in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to determine the skin sensitization potential of HCA following dermal exposure.

Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline [1], the test item was dissolved in N,N-dimethylformamide (abbreviation: DMF). The test item formed an appropriate formulation in this vehicle at concentrations of 25 (w/v)%, therefore it was chosen as vehicle for the test.

The Preliminary Irritation/Toxicity Test was performed in CBA/J Rj mice using two doses (test item concentrations of 25 and 10 (w/v)%) in the selected vehicle. The observations recorded in the preliminary test suggest that the 25 (w/v)% formulation is a suitable dose level for a valid LLNA.

 

In the main assay, sixteen female CBA/J Rj mice were allocated to four groups of four animals each:

- three groups received the appropriate formulation of HCA at concentrations of 25, 10 and 5 (w/v)%,

- the negative control group received DMF,

- the positive control group* received 25% α-Hexylcinnamaldehyde (α-HCA) in DMF.

*To minimise animal use, the positive control group was part of a concurrent study with the same solvent.

 

The solutions of the test item were applied on the dorsal surface of ears of days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (³HTdR) and the values obtained were used to calculate stimulation indices (SI).

No mortality, systemic toxicity or local irritation was observed during the study. No treatment related effects were observed on animal body weights in any other treated groups. The observed clinical signs are summarized in Appendix 3 of attached report.

 

Stimulation index values of the test item were 2.5, 4.4 and 4.2 at treatment concentrations of 25, 10 and 5 (w/v)%, respectively.

 

α-Hexylcinnamaldehyde (25 (w/v)% dissolved in DMF) was used as a positive control to demonstrate the appropriate performance of the assay [1]. A significant lymphoproliferative response (stimulation index value of 16.6) was noted for the positive control chemical and this result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present assay HCA (Batch No.: 00423), tested in a suitable vehicle, was shown to have a sensitization potential (sensitizer) in the Local Lymph Node Assay.

 

[1] OECD Guidelines for Testing of Chemicals No. 429. Skin Sensitisation: Local Lymph Node Assay. Adopted: 22 July 2010.