1-Naphthol, reaction products with formaldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May 2005 to 02 June 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2004

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:

1-Naphthol, reaction products with formaldehyde

EC Number:

607-708-4

Cas Number:

25359-91-5

Molecular formula:

Not applicable - UVCB substance

IUPAC Name:

1-Naphthol, reaction products with formaldehyde

Test material form:

liquid

Details on test material:

- Appearance: dark blue liquid
- Storage: approximately 4 °C in the dark under nitrogen

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/Ca CruBR

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Eight to twelve weeks old.
- Weight at study initiation: 15 to 23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 30 - 70 %
- Air changes: Approximately fifteen changes per hour
- Photoperiod: Twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Undiluted, 50 % or 25 % (v/v).

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: For the purpose of the study the test material was used undiluted and freshly prepared in dimethyl formamide. This vehicle was chosen as it produced the most suitable formulation at the required concentration.
- As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test material a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the undiluted test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded. The bodyweight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.

MAIN STUDY
TEST MATERIAL ADMINISTRATION:
- Groups of five mice were treated with the undiluted test material or the test material at concentrations of 25 % or 50 % v/v in dimethyl formamide. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 μL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
- A further group of five mice received the vehicle alone in the same manner.

3H-METHYL THYMIDINE ADMINISTRATION
- Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR:80μCi/mL, specific activity 2.0 Ci/mmol, Amersham Biosciences UK Ltd) giving a total of 20 μCi to each mouse.

OBSERVATIONS
- Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

TERMINAL PROCEDURES
- Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.
- Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cells suspension was transferred to a 10 mL centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for ten minutes. The pellet was resuspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspended in 3 mL of 5 % Trichloroacetic acid (TCA).
- Determination of 3HTdR Incorporation: After overnight incubation at 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by P-scintillation counting. The "Poly Q TM" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA).

INTERPRETATION OF RESULTS
- The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes from each individual animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index (SI)).
- The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitiser".

Positive control substance(s):

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

Statistics:

Data was processed to give group mean values for dpm and standard deviations where appropriate. Individual and group mean dpm values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett's Multiple Comparison test was used and for non-homogenous datasets Dunnett's T3 Multiple Comparison Method was used.
Probability values (p) are presented as follows:
P <0.001 ***
P <0.01 **
P <0.05 *
P ≥0.05 (not significant)

Results and discussion

Positive control results:

Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of a -Hexylcinnamaldehyde, Tech, 85 % as a solution in acetone/olive oil 4:1 at concentrations of 5, 10 and 25 % v/v:
5 % SI = 2.76 (Negative)
10 % SI = 3.34 (Positive)
25 % SI = 8.91 (Positive)
a -Hexylcinnamaldehyde, Tech, 85 % was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.95

Test group / Remarks:

25 % v/v

Results 2

Parameter:

SI

Value:

0.95

Test group / Remarks:

50 % v/v

Results 3

Parameter:

SI

Value:

1.04

Test group / Remarks:

100 % v/v

Cellular proliferation data / Observations:

PRELIMINARY SCREENING TEST
- No signs of systemic toxicity were noted. Dark blue-coloured staining of the fur and ears was noted one hour post dosing on Days 1, 2 and 3.
- Based on this information the dose levels selected for the main test were 25 or 50 % v/v in dimethyl formamide and 100 %.

MAIN TEST
- Estimation of the Proliferative Response of Lymph Node Cells: A stimulation index of less than 3 was recorded for the three concentrations of the test material (25 or 50 % v/v in dimethyl formamide and 100 %).
- Clinical Observations and Mortality Data: There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Dark blue-coloured staining of the fur and ears was noted in all test animals one hour post dosing on Days 1 to 3.
- Bodyweight: Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Table 1: Mean Group Dpm's and Stimulation Index (SI)

Concentration (% v/v) in Dimethyl Formamide	Mean Dpm/Animal (Standard Deviation)	Stimulation Index (SI)*	Result
Vehicle	1375.81 (± 194.61)	N/A	N/A
25	1313.21 (± 989.36)	0.95	Negative
50	1303.03 (± 609.77)	0.95	Negative
100	1432.57 (± 364.17)	1.04	Negative

*Stimulation Index of 3.0 or greater indicates a positive result

Applicant's summary and conclusion

Interpretation of results:

other: Not classified in accordance with EU Criteria.

Conclusions:

Under the conditions of this study the test material was considered to be a non-sensitiser.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 429 and EU Method B42, under GLP conditions.

The study was performed in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. Following a preliminary screening test, three groups, each of five animals, were treated with 50 μL (25 μL per ear) of the undiluted test material or the test material as a solution in dimethyl formamide at concentrations of 25 or 50 % v/v. A further group of five animals was treated with dimethyl formamide alone.

The Stimulation Index (SI) expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group were 0.95, 0.95 and 1.04 for the 25, 50 and 100 % (v/v) test material concentration groups, respectively.

Under the conditions of this study the test material was considered to be a non-sensitiser.