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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 23 June 2020 to 06 November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study was being performed with vertebrate animals because the chemical nature of the test item was not compatible with the available in vitro alternative tests. The in vitro testing was considered not to be technically feasible.
Indeed, 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

impurity 1
Reference substance name:
Total unspecified impurities
IUPAC Name:
Total unspecified impurities
Constituent 1
Reference substance name:
Cobalt Nickel Manganese Oxide
Cas Number:
37348-84-8
Molecular formula:
Ni1-x-yMnxCoyOz with Me=Ni+Mn+Co=1 and Ni/Me= 0.45-0.98 Mn/Me= 0.01-0.35 Co/Me= 0.01-0.35 O/Me= 1.00-1.33
IUPAC Name:
Cobalt Nickel Manganese Oxide
Test material form:
solid
Specific details on test material used for the study:
Batch/Lot number: PVX-144A PVX14
Description: Grey Powder
Purity: 100% (7.44% Co ; 6.99% Mn ; 59.67% Ni)
Manufacturer: Umicore
Expiry date: 30 April 2021
Storage conditions: Controlled room temperature (15-25 °C, ≤70% relative humidity). Protected from light and humidity (store in a tightly closed container).
The Certificate of Analysis is attached in Appendix 1 of the study report.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo, RMS B.V. Postbus 553, 5800 AN Venra , Netherland
- Females nulliparous and non-pregnant: yes
- The health status of the animals assigned to study were verified by the clinical Veterinarian.
- Age at study initiation: Young adults, 12 weeks old (age-matched, within one week)
- Weight at study initiation: 18.5 – 20.8 grams (the weight variation in animals in the study did not exceed ± 20 % of the mean weight)
Note: In the Preliminary Experiment mice of 8 weeks of age (18.3-21.0 g) were used.
- Housing: Group caging (which is the standard procedure for small rodent studies, following AAALAC recommendations, to allow social interaction) during the observation period and individual caging in the radioactive proliferation test phase in the main experiment. Cages: Type II. polypropylene/ polycarbonate
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: yes. During the acclimation period of at least 20 days, the animals were kept under the same controlled environment conditions as during the experimental period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1 – 28.1°C
- Humidity (%): 30 – 76%
- Air changes (per hr): 15-20 air exchange/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
50, 25, 10 % (w/v)
No. of animals per dose:
4/dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility:
The following standard OECD No. 429 vehicles were assessed: Acetone: Olive oil 4:1 (v/v) mixture, N,N-dimethylformamide, Methyl ethyl ketone (MEK), Propylene glycol, Dimethyl sulfoxide (DMSO) and 1% aqueous Pluronic® PE9200.
The test item did not dissolve in any vehicle at concentration of 100% (w/v), the formulations were quickly settling dispersions. At concentration of 50% (w/v) the formulations with Acetone Olive oil 4:1 (v/v) mixture, N,N-dimethylformamide, Methyl ethyl ketone (MEK) were quickly settling dispersions. The formulation with Propylene glycol (PG) was slowly settling black dispersion which was considered appropriate with continuous mixing by visual inspection. Taking into account the test item characteristics and the study requirements, PG was selected as the vehicle for this study.

The Preliminary Irritation/Toxicity Test was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 50% and 25% (w/v) in PG. 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.
- Irritation: yes
- Systemic toxicity: yes
- Ear thickness measurements: yes
- Erythema scores: yes
During the Preliminary Irritation / Toxicity Test no mortality or clinical signs were observed. Test item residue or a minimal amount of test item residue was observed on the ears of the 50% (w/v) group from Day 1 to Day 6 and 25% (w/v) group from Day 1 to Day 5.
Marked body weight loss (>5% reduction of body weight) was observed in one animal (-10%) in the 50% (w/v) group, however the other animal gained weight (13.7%) and it was considered as an individual case and not test item related effect. No marked body weight loss was observed in average body weight in any dose group in the preliminary test.
Ear thickness of the animals was measured using a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after euthanasia of the experimental animals on Day 6.
No increased ear thickness value (>25%) was detected. Ear punch weights of the animals were within the historical control range.
The draining auricular lymph nodes of the animals were visually examined: they were normal in both dose groups (subjective judgement by analogy with observations of former experiments).
The 50% (w/v) dose group was considered acceptable and was therefore selected as highest dose for the main test.
No ear thickness measurements or ear punch weight determination was required in the main test.

MAIN STUDY
Mortality
Animals were inspected for signs of morbidity and mortality at least once daily.
The principles and criteria summarized in the OECD Humane Endpoints Guidance Document No. 19 were taken into consideration.

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.

Body weights
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
3HTdR

Evaluation 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.
Interpretation results:
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.

Acceptability of te test:
The Local Lymph Node Assay is considered valid if it meets the following criteria:
-the DPN value of the negative (vehicle) control group falls within the range of historical laboratory control data,
-the positive control substance produces a significant lymphoproliferative response (SI>3),
-the positive control is chosen such that it does not cause excessive skin irritation or systemic toxicity and the induction is reproducible but not excessive (i.e. SI>20),
-each treated and control group includes at least 4 animals,
-the test item does not cause serious systemic or local toxicity.

ANIMAL ASSIGNMENT AND TREATMENT
The animals were assigned to their respective dose groups by randomisation based on body weights. It was checked that all animals were within 20% of the overall mean at the start of the study. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; computer software (PROVANTIS v.9.3) was used in order to verify homogeneity/variation among/within groups.
- 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.


TREATMENT PREPARATION AND ADMINISTRATION:
Test item was be freshly dissolved in applicable solvent to obtain appropriate concentrations shortly before application to mice and was considered to be stable for this short period. The applicable doses were based on the results of the Preliminary Irritation/Toxicity Test (see section 10.1). The test item was weighed, and formulations prepared daily on a weight: volume basis as % (w/v) by the Pharmacy of Charles River Laboratories Hungary Kft.
As agreed with the Sponsor, no correction for purity of the test item was applied during formulation.
Analytical determination of the test item concentration, stability and homogeneity was not be performed because of the character and the short period of study.
The formulations were checked for visible homogeneity and physical stability before the start of the preliminary experiment. They were made shortly before application to mice and were considered to be stable for this short period.
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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistical evaluation was performed

Results and discussion

Positive control results:
HCA 25%

Body weight
Animal Number Identity Number Initial Body weight (g) Terminal Body Weight* (g) Change# (%)
9968 17 19.2 18.8 -2.1
9970 18 18.8 18.8 0.0
9985 19 20.7 20.7 0.0
9982 20 20.3 20.5 1.0
Mean 19.8 19.7 -0.3
Notes:
*: Terminal body weights were measured on Day 6.
#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

Proliferation Assay
The appearance of the lymph nodes was larger than normal in the positive control groups.
Measured DPM/group: 35364
DPM: 35328.0
No. of Nodes: 8
DPN: 4416.0
Stimulation Index Values: 10.3

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
50% (w/v)
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
25% (w/v)
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
10% (w/v)
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Negative control (PG)
Key result
Parameter:
SI
Value:
10.3
Test group / Remarks:
Positive control (25% HCA)
Cellular proliferation data / Observations:
The test item was powder, which was formulated in PG. 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 not 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 pNMC oxide (8:1:1) is a non-sensitizer. The size of lymph nodes was in good correlation with this conclusion.

Reliability of the test
The result of the positive control substance α-Hexylcinnamaldehyde (HCA) was used to demonstrate the appropriate performance of the assay [1]. The positive control substance was examined at a concentration of 25% (w/v) in the relevant vehicle (PG) using CBA/CaOlaHsd 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 10.3) 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.
Historical control data for the positive and negative control substances are made available in appendixof the report.

Any other information on results incl. tables

Proliferation Assay

The appearance of the lymph nodes was normal in the 50%, 25% and 10% (w/v) test and negative control groups and larger than normal in the positive control groups.

 

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)

36*

-

-

-

-

Negative control (PG)

3479

3443.0

8

430.4

1.0

50% (w/v)

4305

4269.0

8

533.6

1.2

25% (w/v)

3669

3633.0

8

454.1

1.1

10% (w/v)

3377

3341.0

8

417.6

1.0

Positive control (25% HCA)

35364

35328.0

8

4416.0

10.3

Note: Total DPM = Measured DPM – Background DPM

DPN = (Measured DPM – Background DPM value of TCA) / Number of nodes

*: The background values were 35 and 37.

 

The SI values were 1.2, 1.1, 1.0 at concentrations of 50, 25 and 10% (w/v), respectively.

 

INTERPRETATION OF THE OBSERVATIONS

The test item was powder, which was formulated in PG. 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 not 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 Nickel Cobalt Manganese oxide (8:1:1) is a non-sensitizer. The size of lymph nodes was in good correlation with this conclusion.

 

CLINICAL OBSERVATIONS

There was no mortality or clinical signs observed during the main assay. Test item residue or a minimal amount of test item residue was observed on the ears of the 50% (w/v) group from Day 1 to Day 6 and minimal amount of test item residue of the, 25 and 10% (w/v) group from Day 1 to Day 6.

 

Group

Animal No.

CLINICAL OBSERVATIONS

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

Negative control

(PG)

9986

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

9983

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

9977

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

9979

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

Nickel Cobalt Manganese oxide (8:1:1) 50% (w/v) in PG

9980

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0*

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0*

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0*

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

9976

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0*

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0*

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0*

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

9973

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0*

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0*

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0*

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

9971

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0*

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0*

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0*

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Nickel Cobalt Manganese oxide (8:1:1) 25% (w/v) in PG

9978

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

9984

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

9974

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

9981

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

 

Group

Animal No.

CLINICAL OBSERVATIONS

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6

Nickel Cobalt Manganese oxide (8:1:1) 10% (w/v) in PG

9969

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

9987

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

9975

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

9972

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0**

BT: symptom-free, ES: 0**
AT: symptom-free, ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Symptom-free,
ES: 0**

Positive control

(25% (w/v) HCA

in PG

9968

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

9970

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

9985

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

9982

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

BT: symptom-free, ES: 0
AT: symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

Symptom-free, ES: 0

Notes:

1.              BT: before treatment, AT: after treatment

2.              ES: Erythema score (0 – No erythema)

3.             * Test item residue

4.             ** Minimal amount of test item residue

 

BODY WEIGHT

Marked decrease (>5%) of the body weight was observed in only one animal in the 50% (w/v) group. No marked decrease was observed in any other animals and no test item related effect was noted on body weights. The average body weight range was within the acceptable range of all groups.

 

Individual Body Weights for all Animals with Group Means

 

Animal Number

Identity Number

Test Group Name

Initial Body weight (g)

Terminal Body Weight* (g)

Change#(%)

 
 

9986

1

Negative (vehicle) control
PG

18.8

20.0

6.4

 

9983

2

20.0

21.3

6.5

 

9977

3

20.6

20.1

-2.4

 

9979

4

19.6

20.3

3.6

 

 

 

Mean

19.8

20.4

3.5

 

9980

5

Nickel Cobalt Manganese oxide (8:1:1) 50% (w/v) in PG

20.2

20.2

0.0

 

9976

6

18.5

18.4

-0.5

 

9973

7

20.8

19.2

-7.7

 

9971

8

19.0

18.7

-1.6

 

 

 

Mean

19.6

19.1

-2.5

 

9978

9

Nickel Cobalt Manganese oxide (8:1:1) 25% (w/v) in PG

20.6

20.0

-2.9

 

9984

10

18.6

19.6

5.4

 

9974

11

19.4

18.9

-2.6

 

9981

12

20.3

19.9

-2.0

 

 

 

Mean

19.7

19.6

-0.5

 

 

Animal Number

Identity Number

Test Group Name

Initial Body weight (g)

Terminal Body Weight* (g)

Change#(%)

 
 

9969

13

Nickel Cobalt Manganese oxide (8:1:1) 10% (w/v) in
PG

19.0

19.2

1.1

 

9987

14

20.7

22.0

6.3

 

9975

15

19.2

19.9

3.6

 

9972

16

20.2

19.8

-2.0

 
   

Mean

19.8

20.2

2.2

 

9968

17

Positive control
25 (w/v) % HCA in PG

19.2

18.8

-2.1

 

9970

18

18.8

18.8

0.0

 

9985

19

20.7

20.7

0.0

 

9982

20

20.3

20.5

1.0

 
   

Mean

19.8

19.7

-0.3

 

 Notes:

*: Terminal body weights were measured on Day 6.

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

 

RESULTS OF THE PRELIMINARY IRRITATION / TOXICIYT TEST

Individual Body Weights for all Animals with Group Means (Preliminary Irritation/Toxicity Test)

Animal

Number

Identity

Number

Test Group

Name

Initial Body

Weight (g)

Terminal Body

Weight* (g)

Change#

(%)

9844

1

50% (w/v)

21.0

18.9

-10.0

9845

2

50% (w/v)

18.3

20.8

13.7

 

 

Mean

19.7

19.9

1.0

9846

3

25% (w/v)

21.0

20.4

-2.9

9843

4

25% (w/v)

18.7

18.7

0.0

 

 

Mean

19.9

19.6

-1.5

Notes:

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

2.     *: Terminal body weights were measured on Day 6.

 

Individual Ear Thickness for all Animals(Preliminary Irritation/Toxicity Test)

Animal

Number

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)

Right

Left

Right

Left

Right

Left

9844

1

50% (w/v)

0.22

0.23

0.24

0.25

0.23

0.24

14.8

9845

2

50% (w/v)

0.23

0.22

0.24

0.24

0.23

0.23

15.2

9846

3

25% (w/v)

0.22

0.22

0.23

0.24

0.23

0.23

15.1

9843

4

25% (w/v)

0.22

0.22

0.24

0.25

0.24

0.23

13.9

Notes:

1.      *:In case of ear thickness values, irritant response is considered when result is ≥25% above the Day 1 value.

2.      **:Historical control range: 12.50-21.30 mg. Positive response is over 26.63 mg (≥25%).

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
no sensitisation potential (non-sensitizer)
Conclusions:
In conclusion, under the conditions of the present assay, pNMC oxide (8:1:1), tested in a suitable vehicle (PG), was shown to have no sensitisation potential (non-sensitizer) in the Local Lymph Node Assay.
The study result triggers the following classification/labelling:
- Regulation (EC) No 1272/2008 (CLP): Not classified
- GHS (rev. 8) 2019: Not classified
Executive summary:

The object of this study was to determine the skin sensitisation potential of pNMC oxide (8:1:1) following dermal exposure in mice. The study was being performed with vertebrate animals because the chemical nature of the test item was not compatible with the available in vitro alternative tests. The in vitro testing was considered not to be technically feasible. In this respect, it is important to note that with the adoption of Commission Regulation (EU) 2016/1688 on September 20th, 2016, the testing strategy for the assessment of the skin sensitization 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 propylene glycol (PG). The 50% (w/v) formulation was the highest concentration which was suitable for the preliminary test.

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

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

  1. three groups of animals received the test item at 50, 25 and 10% (w/v, formulated in PG) concentrations,
  2.  a negative control group received the vehicle (PG) only,
  3.  a positive control group received 25% (w/v) α-Hexylcinnamaldehyde (HCA), formulated in PG.

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 or clinical signs observed during the main assay.Test item residue or a minimal amount of test item residue was observed on the ears of the 50% (w/v) group from Day 1 to Day 6 and minimal amount of test item residue of the, 25 and 10% (w/v) group from Day 1 to Day 6.

The average body weight range was within the acceptable range of all groups. The SI values were 1.2, 1.1 and 1.0 at concentrations of 50%, 25% and 10% (w/v), respectively.

The DPN values of the negative control group was in line with historical control data. The SI value for the positive control substance 25% (w/v) α-Hexylcinnamaldehyde (HCA), formulated in the same vehicle as the test item (SI=10.3) demonstrated the appropriate performance of the assay, therefore confirmed the validity of the assay.

In conclusion, under the conditions of the present assay, pNMC oxide (8:1:1), tested in a suitable vehicle (PG), was shown to have no sensitizatonp potential (non-sensitizer) in the Local Lymph Node Assay. The study result triggers the following classification/labeling:

  • Regulation (EC) No 1272/2008 (CLP): not classified
  • GHS (rev.8) 2019: not classified

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