Reaction mass of 1,4-bis(methoxymethyl)benzene, 1,6-dihydroxynaphtalene and Epichlorohydrin

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 November 2019 to 27 November 2019

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,4-bis(methoxymethyl)benzene, 1,6-dihydroxynaphtalene and Epichlorohydrin

EC Number:

836-437-6

Cas Number:

577978-76-8

Molecular formula:

C40H38O8 to C112H104O20

IUPAC Name:

Reaction mass of 1,4-bis(methoxymethyl)benzene, 1,6-dihydroxynaphtalene and Epichlorohydrin

Test material form:

solid

Details on test material:

Name: CAS 577978-76-8
Chemical Name: Polyglycidyl ether of 1,6-naphthalenediol-pxylyleneglycol (or p-xylyleneglycol dimethylether) condensation polymer
CAS number: CAS 577978-76-8
Batch/Lot number: 0950985
Description: Brown solid
Purity: 100%
Expiry date: 08 February 2020 (as per updated CoA)
Storage conditions: Controlled room temperature (15-25oC, ≤70% relative humidity).
Safety precautions: Enhanced safety precautions above the routine safety precautions (lab coat, gloves, safety glasses, face mask) will be applied considering the supplied safety data sheet to assure personnel health and safety.

Specific details on test material used for the study:

No further details specified in the study report

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaCrl

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/CaCrl mice
Source: Charles River UK Limited, Manston Road, Margate, CT9 4LT Kent, GB.
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD No. 429 guideline, mice of CBA/Ca or CBA/J strain can be used. Females were 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: Young adults, 8 weeks old
(age-matched, within one week)
Body weight range at starting: 18.2 – 20.9 grams (the weight variation in animals in the study did not exceed ± 20 % of the mean weight)
Acclimatisation time: At least 13 days
Note: In the Preliminary Experiment mice of 10 weeks of age (21.4 – 22.1 g) 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 will be provided with glass tunnel-tubes
Cage type: Type II. polypropylene/ polycarbonate
Bedding / Nesting: Bedding and certified nest building material will be available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.7-25.6°C
Relative humidity: 25-73%
Ventilation: 15-20 air exchange/hour
The temperature and relative humidity were continuously monitored and recorded twice every day during the acclimatisation and experimental phases.

Food and feeding
Animals received ssniff SM Rat/Mouse – Breeding and Maintenance, 15 mm, autoclavable “Complete feed for Rats and Mice” produced by ssniff Spezialdiäten GmbH (D-59494 Soest, Germany), and Geldiet Transport (Scientific Animal Food & Engineering, Route de Saint Bris, 89290 Augy, France) 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 bottles, 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-8200 Veszprém, József Attila u. 36., Hungary). Copies of the relevant Certificates of Analysis are retained in the Archive at Charles River Laboratories Hungary Kft.

Bedding and nesting
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. SAFE 3/4 S certified wooden chips produced by J. Rettenmaier & Söhne GmbH + Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Certified nest building material was also provided for animals (SAFE crinklets natural produced by J. Rettenmaier & Söhne GmbH + Co.KG).

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 Charles River Laboratories Hungary Kft.’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.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

A Preliminary Irritation/Toxicity Test was performed in CBA/CaCrl mice using two dose levels (2 animals/dose): 50% and 25% (w/v) in DMF and based on the results, 50% (w/v) dose was selected as top dose for the main test.

In the main assay, twenty-four female CBA/CaCrl mice were allocated to six groups, each group comprised four animals:
- four groups of animals received CAS 577978-76-8 at 50%, 25%, 10% and 5% (w/v, formulated in DMF) concentrations,

No. of animals per dose:

Preliminary toxicity test: 2 animals /dose
Main assay: 4 animals / dose

Details on study design:

Dose Selection and Justification of Dose Selection

The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaCrl mice using two doses (2 animals/dose) at test item concentrations of 50% and 25% (w/v) in DMF. 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 the Preliminary Irritation / Toxicity Test, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.

During the Preliminary Irritation / Toxicity Test no mortality occurred. In the 50% (w/v) group very slight or well-defined erythema and extensive alopecia were observed between Day 2 (after treatment) and Day 6. In the 25 (w/v) group very slight erythema was observed on Day 3 (after treatment). Test item residue or a minimal amount of test item residue was observed on the ears of the 50 and 25% (w/v) groups.

Marked body weight loss (>5% reduction of body weight) was observed only in one animal in the 50% (w/v) dose group in the preliminary test and the mean body weight loss was -5.1%. There was no marked body weight loss in the 25% (w/v) group

Ear thickness of the animals was measured using a thickness gauge on Days 1, 3 and 6. No increased ear thickness value (>25%) was detected. Ear punch weights of the animals of both dose groups, were within the acceptable range.

The draining auricular lymph nodes of the animals were visually examined: they were larger than normal in both dose groups (subjective judgement by analogy with observations of former experiments).

Despite the marked body weight loss in one mouse in the 50% (w/v) group, this concentration was not excluded from the experiment since earlier experience shows that it can appear in a control group as well. Excluding this group from the main test could lead to a false negative result.
Based on these results, 50% (w/v) dose was selected as top dose for the main test and four concentrations was considered appropriate, in order to avoid further animal testing in case the 50% (w/v) proves to be irritant in the main test.

No ear thickness measurements or ear punch weight determination was performed in the main test.

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.

OBSERVATIONS

Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

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 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.

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 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.
Since the test item gave a positive response and data permitted, the EC3 value of the test item was calculated (EC3 means the effective chemical concentration required for SI=3). The calculation of the EC3 value was conducted by log-linear interpolation.

Positive control substance(s):

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

Results and discussion

Positive control results:

The result of the positive control substance α-Hexylcinnamaldehyde (HCA) was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25% (w/v) in the relevant vehicle (DMF) using CBA/CaCrl mice.

No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 6.9) 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 test item treated and control group included 4 animals.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

14.6

Test group / Remarks:

50% (w/v)

Results 2

Key result

Parameter:

SI

Value:

8

Test group / Remarks:

25% (w/v)

Results 3

Key result

Parameter:

SI

Value:

7.3

Test group / Remarks:

10% (w/v)

Results 4

Key result

Parameter:

SI

Value:

5.3

Test group / Remarks:

5% (w/v)

Cellular proliferation data / Observations:

Cell proliferation in the (local) lymph nodes was assessed by measuring disintegrations per minutes after the incorporation of tritiated methyl thymidine (3HTdR) into the lymph nodes and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

Any other information on results incl. tables

CLINICAL OBSERVATION

There was no mortality observed during the main assay. In the 50% (w/v) group very slight erythema and extensive alopecia were observed between Day 3 (before treatment) and Day 6. No clinical signs were observed in any other group. Minimal amount of test item residue was observed on the ears of the 50% (w/v) groups on Day 3 after treatment. No test item residue was observed in any other group.

BODY WEIGHT MEASUREMENT

No test item related effect was noted on body weight.

PROLIFERATION ASSAY

The appearance of the lymph nodes was larger than normal in the 50% (w/v) group and positive control group, slightly larger than normal in the 25% (w/v) group and normal in the negative control group and in the other test item treated dose groups.

The stimulation index values were 14.6, 8.0, 7.3 and 5.3 at concentrations of 50, 25, 10 and 5% (w/v), respectively.

INTERPRETATION OF OBSERVATIONS

The test item was solid, which was applied formulated in DMF. 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. The resulting stimulation indices observed under these exaggerated test conditions were considered to be good evidence that CAS 577978-76-8 is a sensitizer.

The dose response does not allow a reliable extrapolation to the EC3 value, although it appears to be above 2% (calculated EC3 equals to 2.3% suggesting a Cat.1B). To be sure of the classification a further group at 2% test item would be required.

Based on the observed results, the test item needs classification of Category 1 according to the GHS or CLP

Summary of Preliminary Study Data

Concentrations	Physical Formulation	Clinical Observations	Body Weight	Erythema	Ear Thickness	Ear Biopsy weight
50% (w/v)	A	A	E	A	A	A
25% (w/v)	A	A	A	A	A	A

Notes: U=Unacceptable; A=Acceptable; E=Equivocal; NM=Not measured

Experimental Groups and Treatments

Groups	Test item concentration (% w/v)	No. of animals
Negative (vehicle) control (DMF)	---	4
50% (w/v) in DMF	50	4
25% (w/v) in DMF	25	4
10% (w/v) in DMF	10	4
5% (w/v) in DMF	5	4
Positive (25% (w/v) HCA in DMF)	---	4

Individual Body Weights for all Animals with Group Means

Identity Number	Animal Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight* (g)	Change# (%)
1	8880	Negative (vehicle) control (DMF)	20.4	21.3	4.4
2	8891		20.4	21.3	4.4
3	8890		19.0	19.9	4.7
4	8879		18.3	18.9	3.3
		Mean	19.5	20.4	4.2
5	8881	CAS 577978-76-8 50% (w/v) in DMF	20.4	21.2	3.9
6	8899		19.9	20.5	3.0
7	8889		19.3	19.4	0.5
8	8878		19.7	21.0	6.6
		Mean	19.8	20.5	3.5
9	8892	CAS 577978-76-8 25% (w/v) in DMF	20.5	21.0	2.4
10	8893		19.9	20.6	3.5
11	8877		19.5	21.2	8.7
12	8888		18.5	18.7	1.1
		Mean	19.6	20.4	4.0
13	8900	CAS 577978-76-8 10% (w/v) in DMF	20.9	22.0	5.3
14	8894		19.3	18.7	-3.1
15	8883		19.1	19.3	1.0
16	8887		18.3	19.0	3.8
		Mean	19.4	19.8	1.8
17	8895	CAS 577978-76-8 5% (w/v) in DMF	20.4	20.8	2.0
18	8882		19.3	19.8	2.6
19	8886		19.3	19.6	1.6
20	8896		18.2	19.0	4.4
		Mean	19.3	19.8	2.6
21	8885	CAS 577978-76-8 25% (w/v) HCA in DMF	20.1	20.3	1.0
22	8898		20.0	22.0	10.0
23	8897		19.3	20.7	7.3
24	8884		18.2	18.9	3.8
		Mean	19.4	20.5	5.5

Notes:

*: Terminal body weights were measured on Day 6.

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

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Measured DPM/group	DPM	No. of Nodes	DPN	Stimulation Index Values
Background (5 (w/v) % TCA)	34	---	---	---	---
Negative control (DMF)	4097	4063.0	8	507.9	1.0
50% (w/v)	59206	59172.0	8	7396.5	14.6
25% (w/v)	32657	32623.0	8	4077.9	8.0
10% (w/v)	29564	29530.0	8	3691.3	7.3
5% (w/v)	21529	21495.0	8	2686.9	5.3
Positive control (25% HCA)	28158	28124.0	8	3515.5	6.9

 The stimulation index values were 14.6, 8.0, 7.3 and 5.3 at concentrations of 50, 25, 10 and 5% (w/v), respectively.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the conditions of the present assay, CAS 577978-76-8, tested in a suitable vehicle (DMF), was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

Executive summary:

The object of this study was to determine the skin sensitisation potential of CAS 577978-76-8 following dermal exposure in mice. The study was being performed with vertebrate animals because the chemical nature of the test item is not compatible with the available in vitro alternative tests*. The in vitro testing was considered not to be technically feasible.

*IMPORTANT NOTE: With Commission Regulation (EU) 2016/1688 adopted on the 20th September 2016, the testing strategy for the assessment of the skin sensitisation potential of a new substance now includes mandatory in vitro testing before any in vivo testing can be carried out, if the in vitro tests are technically feasible. This applies to all substances to be tested for EU REACH and CLP, but not for other regulatory purposes.

Before the start of this in vivo study, the Sponsor confirmed that existing data was not sufficient for the labelling or for the specific regulatory purpose for skin sensitisation.

The solubility of the test item was examined in a short Preliminary Compatibility Test. The best vehicle taking into account the test item characteristics and the requirements of the relevant OECD guideline was considered to be N,N-dimethylformamide (DMF). The 50% formulation was the highest concentration which was suitable for the preliminary test.

A Preliminary Irritation/Toxicity Test was performed in CBA/CaCrl mice using two dose levels (2 animals/dose): 50% and 25% (w/v) in DMF and based on the results, 50% (w/v) dose was selected as top dose for the main test.

In the main assay, twenty-four female CBA/CaCrl mice were allocated to six groups, each group comprised four animals:

Four groups of animals received CAS 577978-76-8 at 50%, 25%, 10% and 5% (w/v, formulated in DMF) concentrations,

A negative control group received the vehicle (DMF) only,

A positive control group received 25% (w/v) HCA (formulated in DMF).

The formulations were applied to the dorsal surface of the ears of the experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3) and then animals were maintained on study for an additional 3 days. Cell proliferation in the (local) lymph nodes was assessed by measuring disintegrations per minutes after the incorporation of tritiated methyl thymidine (3HTdR) into the lymph nodes and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.

There was no mortality observed during the main assay. In the 50% (w/v) group very slight erythema and extensive alopecia were observed between Day 3 (before treatment) and Day 6. No clinical signs were observed in any other group. Minimal amount of test item residue was observed on the ears of the 50% (w/v) groups on Day 3 after treatment. No test item residue was observed in any other group.

No test item related effect was noted on body weight.

The SI values were 14.6, 8.0, 7.3 and 5.3 at concentrations of 50, 25, 10 and 5% (w/v), respectively.

The dose response does not allow a clear classification as 1A or 1B, only as Cat.1.

The DPN values of the negative control group was in line with historical control data. The SI value for the positive control substance α-Hexylcinnamaldehyde (HCA), formulated in the same vehicle as the test item (SI=6.9) demonstrated the appropriate performance of the assay, therefore confirmed the validity of the assay.

In conclusion, under the conditions of the present assay, CAS 577978-76-8, tested in a suitable vehicle (DMF), was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay.

The study result triggers the following classification/labelling:

Regulation (EC) No 1272/2008 (CLP): Category 1

GHS (rev. 8) 2019: Category 1