Reaction products of 1,4-Benzenedimethanol and 1-naphthol

Endpoint summary

Description of key information

LLNA:

Under the conditions of the present assay SN-475N, tested in a suitable vehicle, was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 November 2012 to 13 November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In-Vivo study carried out as substance is intended for global registration where In-Vivo data is required.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

No further details specified in the study report

Species:

mouse

Strain:

CBA

Remarks:

CBA/J Rj mice

Sex:

female

Details on test animals and environmental conditions:

EXPERIMENTAL ANIMALS
Species and strain: CBA/J Rj mice
Source: ELEVAGE JANVIER
Route des Chènes Secs B.P. 4105
53940 LE GENEST-ST-ISLE, France
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 8 weeks old (age-matched, within one week)
Body weight range at starting: 20.3-22.6 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 13 days
Note: In the first preliminary experiment, mice of 11 weeks of age (23.8-25.0 grams) were used. In the second preliminary experiment, mice of 9 weeks of age (18.7-20.2 grams) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
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
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.1 – 25.0 °C
Relative humidity: 30 - 81 %
Ventilation: 15-20 air exchange/hour

The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.
Room/Cabinet (non-radioactive phase): 244/4
Room/Cabinet (radioactive phase): 139 – 140

Food and feeding
Animals received ssniff SM R/M-15mm "Autoclavable complete diet for rats and mice – breeding and maintenance" (Batch number: 523 7816, Expiry date: 30 January 2013) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water supply
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 Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at CiToxLAB Hungary Ltd.

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-FASERN Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH & Co.KG (Holzmühle 1, 73494 Rosenberg, Germany) was available to animals during the study.

Identification and randomisation
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of CiToxLAB Hungary 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 using the body weight to verify the homogeneity and variability between the groups.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

The Preliminary Irritation / Toxicity Tests were performed in CBA/J Rj mice using four doses: 100, 50, 25 and 10 (w/v) % in the selected vehicle. Based on the observations recorded in the preliminary test, the 25 (w/v) % was selected as top dose for the main test.

No. of animals per dose:

4 animals / group

Details on study design:

ADMINISTRATION OF THE TEST ITEM
Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Test I. was started according to the Study Plan on CBA/J Rj mice using two doses (2 animals/dose) at test item concentrations of 100 and 50 (w/v) % in AOO. The preliminary experiment was conducted in a similar experimental manner to the main study, but it was terminated on Day 6 and the radioactive proliferation assay was not performed.
In this experiment, no mortality or clinical signs of systemic toxicity were observed. Marked body weight loss (>5%) was observed in the 100 and 50 (w/v) % dose groups. Test item residue was observed on the ears of all animals on Days 1-6. Alopecia and rigid ear were observed in the 100 (w/v) % dose group on Days 2-6; alopecia was observed for animals of the 50 (w/v) % group on Days 2-6 or Days 3-6. Rigid ears were observed in the 50 (w/v) % dose group on Days 2-5.
Ear thickness of the animals was measured using by a thickness gauge on Days 1, 3 and 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals. Increased ear thickness values were detected in the 100 and 50 (w/v) % dose groups indicating excessive local skin irritation. The revealing ear punch weights were also over the limit of the positive response. However, it can not be excluded that test item remaining on the ears may have contributed to the thickness and weight.
Based on these results with body weight loss of >5% (unacceptable for main study) and potential irritation in the 100 and 50 (w/v) % doses, an additional preliminary experiment (Preliminary Irritation/Toxicity Test II.) was performed to allow suitable dose selection using two doses (2 animals/dose) at test item concentrations of 25 and 10 (w/v) % in AOO.
In this experiment, no mortality or clinical signs of systemic toxicity were observed. No marked body weight loss was observed in the 25 and 10 (w/v) % dose groups. Test item residue was observed on the ears of all animals on Days 2-3 in both dose groups. Minimal alopecia was observed in the 25 (w/v) % dose group on Day 6. Increased ear thickness values were detected in the 25 and 10 (w/v) % dose indicating excessive local skin irritation. The revealing ear punch weights were out of the historical control range but did not exceed the limit of positive response (upper limit of historical control range + 25%).
The draining auricular lymph nodes of the animals were visually examined in both preliminary experiments: they were considered to be larger than normal in all dose groups (subjective judgement by analogy with observations of former experiments).
Based on these results, 100 and 50% doses were considered to be unacceptable for a main study based on the body weight effect (loss of >5%). At 25% there was an indication of local irritation, but since the possibility of test item remaining on the ears can not be excluded, the 25% level was included in the main study. It is sure that a 10 (w/v) % concentration was a suitable concentration in the main test; hence an additional dose group (a total of four) was used in the main experiment to ensure that there will be three acceptable dose groups. Additionally, ear thickness of the experimental animals in the main test were measured by using a thickness gauge on Days 1 and 6 and by ear punch weight determinations, which was performed on Day 6 after the animals are humanely killed. The ear punches were also retained in formalin in case the Sponsor would like to have histological examination for irritation (at additional cost).

Topical application
During the study, animals were topically dosed with 25 μL of the appropriate formulation using a pipette on the dorsal surface of each ear. 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.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (phosphate buffered saline) containing approximately 20 μCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels. Once removed, the nodes of mice from each test group was pooled and collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of pooled lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). Pooled LNCs were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C.
After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 (w/v) % TCA solution was added to the tubes for precipitation of macromolecules.
After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4oC), and supernatants were removed. Pellets were resuspended in 1 mL of 5 (w/v) % TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).
The β-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 (w/v) % TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

Positive control substance(s):

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

Statistics:

Not specified

Positive control results:

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay [1]. A significant lymphoproliferative response (stimulation index value of 11.5) was noted for the positive control chemical, this result confirmed the validity of the assay.

Key result

Parameter:

SI

Value:

22.2

Test group / Remarks:

2.5%

Key result

Parameter:

SI

Value:

50.8

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

119.6

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

133.6

Test group / Remarks:

25%

Cellular proliferation data / Observations:

The appearance of the lymph nodes was normal in the negative (vehicle) control group. Larger than normal lymph nodes were observed in all test item treated and positive control groups.

CLINICAL OBSERVATION

No mortality or signs of systemic toxicity were observed during the study. Alopecia was observed in the 25 % (w/v) group on Days 2-6, and in the 10 % (w/v) group on Day 6. Rigid ears were observed in the 25 % (w/v) group on Days 3-6. There were no indications of any irritancy at the site of application.

BODY WEIGHT MEASUREMENT

No marked body weight loss (>5%) was detected for the test item treated animals except of two animals in the 5 (w/v) % group (6.8 and 5.4%) and one animal in the 2.5 (w/v) % group (5.3%). Similar value was observed for one positive control animal (5.5%). Therefore, it was considered as no treatment related effect.

EAR THICKNESS MEASUREMENTS

Increased thickness and biopsy weight values were observed in the 25 and 10 % dose groups and for some animal in the 5 % dose group. Increased biopsy weights were also detected 25 and 10 % dose groups, although the values did not exceed the limit of positive response (upper limit of historical control range + 25%).

PROLIFERATION ASSAY

The appearance of the lymph nodes was normal in the negative (vehicle) control group. Larger than normal lymph nodes were observed in all test item treated and positive control groups.

No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A significant lymphoproliferative response (stimulation index value of 11.5) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.

Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range. Each treated and control groups included 4 animals.

Results of the Preliminary Irritation/ Toxicity Test I.

Individual Body Weights for all Animals with Group Means

Animal ID	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body weight* (g)	Changes # (%)
298	1	100%	24.1	22.7	-5.8
308	2	100%	23.8	227.	-4.6
Mean			24.0	22.7	-5.2
303	3	50%	25.0	23.4	-6.4
307	4	50%	23.8	22.8	-4.2
Mean			24.4	23.1	-5.3

 

Notes:

1. *Terminal body weights were measured on Day 6

2. # := (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

Individual Ear Thickness for all Animals

Animal ID	Identity Number	Test Group Name	Ear Thickness on Day 1 (mm)	Ear Thickness on Day 3 (mm)	Ear Thickness on Day 6 (mm)	Biopsy weight on Day 6 (mg)
298	1	100 (w/v)%	0.20 / 0.22	0.51 / 0.43	---- / 0.50	23.25
308	2	100 (w/v)%	0.21 / 0.23	0.54 / 0.64	0.78/ 0.48	31.34
303	3	50 (w/v)%	0.21 / 0.22	0.34 / 0.42	0.66/ 0.31	29.17
307	4	50 (w/v)%	0.20 / 0.20	0.37 / 0.33	0.51/ 0.48	32.26

 

Notes:

1. Ear thickness measurements: (data of right ear) / (data of left ear)

2. For biopsy, the average weight range is 13.2-21.4 mg. Positive response is over 26.8 mg. However, it could not be excluded that test item remaining on the ears may have contributed to the thickness and weight at higher concentrations.

Summarized Clinical Observations

Period	Group	Identity No.	Animal ID	Clinical observations
Day 1	100 (w/v)%	1	298	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
		2	308	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	50 (w/v)%	3	303	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
		4	307	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
Day 2	100 (w/v)%	1	298	Before treatment: alopecia, rigid ear*, ES: n.d. After treatment: alopecia, rigid ear*, ES: n.d.
		2	308	Before treatment: alopecia, rigid ear*, ES: n.d. After treatment: alopecia, rigid ear*, ES: n.d.
	50 (w/v)%	3	303	Before treatment: alopecia, slightly rigid ear**, ES: n.d. After treatment: alopecia, slightly rigid ear*, ES: n.d.
		4	307	Before treatment: symptom-free **, ES: n.d. After treatment: slightly rigid ear*, ES: n.d.
Day 3	100 (w/v)%	1	298	Before treatment: alopecia, rigid ear*, ES: n.d. After treatment: alopecia, rigid ear*, ES: n.d.
		2	308	Before treatment: alopecia, rigid ear*, ES: n.d. After treatment: alopecia, rigid ear*, ES: n.d.
	50 (w/v)%	3	303	Before treatment: alopecia, rigid ear*, ES: n. d. After treatment: alopecia, rigid ear*, ES: n. d.
		4	307	Before treatment: alopecia, slightly rigid ear*, ES: n. d. After treatment: alopecia, rigid ear*, ES: n. d.
Day 4	100 (w/v)%	1	298	alopecia, rigid ear*, ES: n. d.
		2	308	alopecia, rigid ear*, ES: n. d.
	50 (w/v)%	3	303	alopecia, slightly rigid ear*, ES: n. d
		4	307	alopecia, slightly rigid ear*, ES: n. d.
Day 5	100 (w/v)%	1	298	alopecia, rigid ear*, ES: n. d.
		2	308	alopecia, rigid ear*, ES: n. d.
	50 (w/v)%	3	303	alopecia, slightly rigid ear*, ES: n. d.
		4	307	alopecia, slightly rigid ear*, ES: n. d.
Day 6	100 (w/v)%	1	298	alopecia, rigid ear*, #, ES: n. d.
		2	308	alopecia, rigid ear*, ES: n. d.
	50 (w/v)%	3	303	alopecia**, ES: 0
		4	307	alopecia**, ES: 0

Notes:

1. The clinical observation of animals on the first day was performed simultaneously with the body weight measurements.

2. ES: Erythema score

3. *: test item residue, **: minimal amount of test item residue

4. n.d. : no data (Due to test item residue, erythema could not be scored at this time point.)

5. #: the right ear of the animal was rigid due to the test item residue

Results of the Preliminary Irritation / Toxicity Test II.

Individual Body Weights for all Animals with Group Means

Animal ID	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body weight* (g)	Changes # (%)
585	1	25%	19.3	19.2	-0.5
596	2	25%	18.7	19.7	5.3
Mean			19.0	19.5	2.4
587	3	10%	20.2	20.4	1.0
601	4	10%	20.0	21.1	5.5
Mean			20.1	20.8	3.2

Notes:

1. *Terminal body weights were measured on Day 6

2. # := (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

Individual Ear Thickness for all Animals

Animal ID	Identity Number	Test Group Name	Ear Thickness on Day 1 (mm)	Ear Thickness on Day 3 (mm)	Ear Thickness on Day 6 (mm)	Biopsy weight on Day 6 (mg)*
585	1	25 (w/v) %	0.22 / 0.22	0.24 / 0.26	0.37 / 0.36	25.56
596	2	25 (w/v) %	0.23 / 0.22	0.25 / 0.26	0.39 / 0.38	26.05
587	3	10 (w/v) %	0.21 / 0.23	0.26 / 0.27	0.28 / 0.28	22.87
601	4	10 (w/v) %	0.24 / 0.22	0.25 / 0.26	0.32 / 0.32	23.87

 

Notes:

1. Ear thickness measurements: (data of right ear) / (data of left ear)

2. For biopsy, the average weight range is 13.2-21.4 mg. Positive response is over 26.8 mg.

Summarized Clinical Observations

Period	Group	Identity No.	Animal ID	Clinical observations
Day 1	25 (w/v)%	1	585	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
		2	596	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	10 (w/v)%	3	587	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
		4	601	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
Day 2	25 (w/v)%	1	585	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: n.d.
		2	596	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: n.d.
	10 (w/v)%	3	587	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: n.d.
		4	601	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: n.d.
Day 3	25 (w/v)%	1	585	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: n.d.
		2	596	Before treatment: symptom-free, ES: 0 After treatment: symptom-free*, ES: n.d.
	10 (w/v)%	3	587	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: n.d.
		4	601	Before treatment: symptom-free, ES: 0 After treatment: symptom-free**, ES: n.d.
Day 4	25 (w/v)%	1	585	symptom-free, ES: 0
		2	596	symptom-free, ES: 0
	10 (w/v)%	3	587	symptom-free, ES: 0
		4	601	symptom-free, ES: 0
Day 5	25 (w/v)%	1	585	symptom-free, ES: 0
		2	596	symptom-free, ES: 0
	10 (w/v)%	3	587	symptom-free, ES: 0
		4	601	symptom-free, ES: 0
Day 6	25 (w/v)%	1	585	alopecia, ES: 0
		2	596	alopecia, ES: 0
	10 (w/v)%	3	587	symptom-free, ES: 0
		4	601	symptom-free, ES: 0

 

Notes:

1. The clinical observation of animals on the first day was performed simultaneously with the body weight measurements.

2. ES: Erythema score

3. *: test item residue, **: minimal amount of test item residue

4. n. d.: no data (Due to test item residue, erythema could not be scored at this time point.)

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the conditions of the present assay SN-475N, tested in a suitable vehicle, was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to determine the skin sensitisation potential of SN-475N following dermal exposure. The study was performed with vertebrate animals as no in vitro alternative is available.

Based on the results of the Preliminary Compatibility Test and on the recommendations of the OECD Guideline [1], the test item was tested for formulation compatibility in AOO (acetone:olive oil 4:1 (v:v) mixture). The highest achievable concentration was 100 (w/v) %.

The Preliminary Irritation / Toxicity Tests were performed in CBA/J Rj mice using four doses: 100, 50, 25 and 10 (w/v) % in the selected vehicle. Based on the observations recorded in the preliminary test, the 25 (w/v) % was selected as top dose for the main test.

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

- four groups received SN-475N (formulated in AOO) at 25, 10, 5 and 2.5 (w/v)% concentrations,

- the negative control group received the vehicle (AOO),

- the positive control group received 25 (w/v) % HCA (dissolved in AOO).

The test item solutions 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 or systemic clinical signs were observed during the study. No marked body weight loss was detected for test item treated animals. Alopecia was observed in the 25 % (w/v) group on Days 2-6, and in the 10 % (w/v) group on Day 6. Rigid ears were observed in the 25 % (w/v) group on Days 3-6.

The observed stimulation index values were 133.6, 119.6, 50.8 and 22.2 at concentrations of 25, 10, 5 and 2.5 (w/v) %, respectively.

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay [1]. A significant lymphoproliferative response (stimulation index value of 11.5) was noted for the positive control chemical, this result confirmed the validity of the assay.

In conclusion, under the conditions of the present assay SN-475N, tested in a suitable vehicle, was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Justification for type of information:

In-Vivo study carried out as substance is intended for global registration where In-Vivo data is required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available