1-Naphthol, reaction products with formaldehyde

Endpoint summary

Description of key information

LLNA, Mullaney (2006)

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

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:

09 May 2005 to 02 June 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2002

Deviations:

no

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)

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.

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)

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.

Parameter:

SI

Value:

0.95

Test group / Remarks:

25 % v/v

Parameter:

SI

Value:

0.95

Test group / Remarks:

50 % v/v

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

KeratinoSens™ Assay, Woutersen (2018)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442D, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to evaluate the ability of the test material to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens™ assay.

A correction factor of 5 was used to correct for the purity of the test material (20 %). The test material was dissolved Milli-Q water at 40 mg/mL. From this stock 11 spike solutions in Milli-Q water were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.20 - 400 µg/mL (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. The test material precipitated at test concentrations of 100 µg/mL and upwards. Two independent experiments were performed.

Both experiments passed the acceptance criteria:

- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. 

- The EC1.5 of the positive control was between 5 and 125 µM (55 µM and 69 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.56-fold and 2.54-fold in experiment 1 and 2, respectively).

- The average coefficient of variation of the luminescence reading for the vehicle (negative) control Milli-Q water was below 20 % (5.7 and 5.3 % in experiment 1 and 2, respectively).

- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20 % (9.8 and 5.7 % in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

The test material showed toxicity (IC30 values of 14 and 17 µg/mL in experiment 1 and 2, respectively, and IC50 values of 20 µg/mL in both experiments). An induction of the luciferase activity (EC1.5 values of 4.0 and 3.3 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 4.85-fold and 3.13-fold in experiment 1 and 2 respectively. 

Under the conditions of this study the test material is classified as positive in the KeratinoSens™ assay since positive results (>1.5-fold induction) were observed at test concentrations < 200 µg/mL with a cell viability of >70 % compared to the vehicle control.

WoE Justification, Pelgrom (2018)

The objective of this study was to evaluate whether sufficient information is available to meet the information requirements for skin sensitisation of Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the relevant classification in accordance with Regulation (EC) No 1272/2008 (CLP) and related amendments. A weight of evidence approach according to Annex XI, sections 1.2 -1.5, to the REACH Regulation is used.

The design of this study is based on the following ECHA guidance: Guidance on information requirements and chemical safety assessment Chapter R.7a Endpoint specific guidance v.6.0 July 2017, paragraph 7.3.

For UVCB substances only two in vitro tests from the AOP are available, as the DPRA is not suitable and also no reliable QSAR prediction can be made. In the KeratinoSens™ assay a positive result was measured with a 20 % solution of the test material in water, with a >1.5-fold induction in two experiments (4.85 and 3.13 in experiment 1 and 2, respectively).

Based on the positive KeratinoSens™ assay performed, no definite conclusion on skin sensitization properties and potency can be drawn, as no single in vitro study is considered reliable for a final conclusion on the endpoint skin sensitization. Performing a second in vitro study also will not result in a definite overall conclusion, as with a negative result no overall conclusion on skin sensitizing potential can be drawn, whereas with a positive or inconclusive result no information on the potency is available. So irrespective of the results of the second in vitro test further testing is needed. In the absence of a scientific reason for a second in vitro test to come to a definite conclusion on the endpoint skin sensitization, it is recommended to perform an in vivo test (LLNA, in the absence of an acceptable alternative) to determine skin sensitization properties and potential.

LLNA, Mullaney (2006)

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 awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

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.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin sensitisation.