Biphenyl-4-ol

Endpoint summary

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:

Start of preliminary test: 04 May 2011; Start of experiment: 18 May 2011; End of experiment: 24 May 2011; Draft report: 15 June 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: OECD Guideline and GLP compliant study.

Justification for data waiving:

other:

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

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)

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: Group caging/mice were provided with glass tunnel-tubes (Cage type: Type II. polypropylene/ polycarbonate; Bedding: Bedding was available to animals during the study. Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Sohne GmbH + Co. KG (Holzmühle 1, 73494 Rosenberg, Germany) 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 6.00 pm.

Room/Cabinet (non-radioactive phase): 244/3
Room/Cabinet (radioactive phase): 139 - 140

Route:

epicutaneous, open

Vehicle:

other: DMF

Concentration / amount:

See attached report

Route:

epicutaneous, open

Vehicle:

other: DMF

Concentration / amount:

See attached report

No. of animals per dose:

4

Positive control substance(s):

yes

Vehicle:

other: N,N-dimethylformamide

Concentration:

50, 25 and 10 (w/v)%

No. of animals per dose:

4 animals/treatment group

Details on study design:

RANGE FINDING TESTS:
- See Appendix of attached report for results of preliminary irritation/toxicity test.
- The Preliminary Irritation/Toxicity Test was performed in CBA/J Rj mice using two doses (test item concentrations of 50 and 25 (w/v)%) in the selected vehicle. The observations recorded in the preliminary test suggest that the 50 (w/v)% formulation is a suitable dose level for a valid LLNA.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- 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.

- 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
- Dose Selection and Justification of Dose Selection: A Preliminary Irritation/Toxicity Test was performed on CBA/J Rj mice using two doses, at test item concentrations of 50 and 25 (w/v)%, respectively. This preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 with a body weight measurement. Radioactive proliferation assay was not performed. During the Preliminary Irritation / Toxicity Test no mortality, systemic toxicity or local irritation were observed (score of erythema was 0 during the observation time). No treatment related effect on body weights was observed in the treated groups. Ear thickness was measured by using a thickness gauge on Days 1, 3 and 6 and the weight of an ear punch on Day 6. The observations recorded in this preliminary test suggest that the formulations, the application of the material and the local effects on the animal are acceptable for a valid LLNA. The observations are summarized in Appendix 2 of attached report. The experimental groups and dose levels are summarized in Table 2 of the attached report.

- 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%

Statistics:

EVALUATION OF THE RESULTS
- DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (‘’DPM’’). The average of the two measured DPM values of 5 (w/v)% TCA solutions was used as the background DPM value. The results were expressed as ‘’DPN’’ (DPM divided by the number of lymph nodes) following the industry standard for data presentation. Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

Positive control results:

- Mean initial body weight: 22.0 g
- Mean terminal body weight: 22.6 g
- Measured DPM/group: Positive control 25% α-HCA in DMF: 14806
- Group DPM: Positive control 25% -HCA in DMF: 14764.5
- No. of Nodes: 8
- DPN: 1845.6
- Stimulation Index Values: 15.8

Parameter:

SI

Remarks on result:

other: Negative control DMF: 1.0 P-PP 50 (w/v)% in DMF: 6.5 P-PP 25 (w/v)% in DMF: 4.6 P-PP 10 (w/v)% in DMF: 3.3 Positive control 25% -HCA in DMF: 15.8

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: 977 - P-PP 50 (w/v)% in DMF: 6143 - P-PP 25 (w/v)% in DMF: 4324 - P-PP 10 (w/v)% in DMF: 3097 - Positive control 25% α-HCA in DMF: 14806 Group DPM: - Negative control DMF 935.5 - P-PP 50 (w/v)% in DMF: 6101.5 - P-PP 25 (w/v)% in DMF: 4282.5 - P-PP 10 (w/v)% in DMF: 3055.5 - Positive control 25% -HCA in DMF: 14764.5

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.

 

INTERPRETATION OF OBSERVATIONS: 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 P-PP at concentrations of 50, 25 and 10 (w/v)%. Stimulation index values of the test item were 6.5, 4.6 and 3.3 at treatment concentrations of 50, 25 and 10 (w/v)%, respectively. The stimulation index values were compatible with a biological dose- related response showing a clear positive response (Figure 1 of attached report).

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, under the conditions of the present assay P-PP (Batch No.: 80832), tested in a suitable vehicle, was shown to have a sensitization potential (sensitizer) in the Local Lymph Node Assay. An EC3 of 5.717% was calculated by the applicant.

Executive summary:

The aim of this study was to determine the skin sensitization potential of P-PP 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 inN,N-dimethylformamide (abbreviation: DMF). The test item formed an appropriate formulation in this vehicle at concentrations of 50 (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 50 and 25 (w/v)%) in the selected vehicle. The observations recorded in the preliminary test suggest that the 50 (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 P-PP at concentrations of 50, 25 and 10 (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 experimental animals (25 µl/ear) for three consecutive 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 6.5, 4.6 and 3.3 at treatment concentrations of 50, 25 and 10 (w/v)%, respectively. Although an EC3 value was not calculated and included in the report, based on the available data and regression analysis an EC3 is calculated to be 5.717 % w/v which is equivalent to 1,429 µg/cm² following relevant ECHA guidance R.8 (EC3 [%] x 250 [µg/cm² / %] = EC3 [µg/cm²]).

 

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 15.8) 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 P-PP (Batch No.: 80832), 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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Migrated from Short description of key information:
In conclusion, under the conditions of the present assay P-PP (Batch No.: 80832), tested in a suitable vehicle, was shown to have a sensitization potential (sensitizer) in the Local Lymph Node Assay.