isliFe

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 May 2019 - 28 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Test material form:

solid

Specific details on test material used for the study:

Batch TROD7BB06

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Remarks:

CBA/JRj

Sex:

female

Details on test animals and environmental conditions:

Female CBA/J (CBA/JRj) strain mice (SPF caw) were supplied by Elevage Janvier Labs (F-53941 Le Genest Saint Isle). On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant.
After an acclimatisation period of at least five days under stabling and nutritional conditions identical to those of the test, the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card.
At the start of the main study the animals were 8 weeks old.
The animals were weighed at the beginning and at the end of the study (see table 3).

Housing
The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
The temperature and relative humidity were controlled to remain within target ranges of 19°C to 25°C and 30% to 70%, respectively.
The rate of air exchange was at least ten changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (07.00 to 19.00) and twelve hours darkness.
The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Food and drink
The drinking water (tap water from public distribution system) and food (ENVIGO 2016) were supplied ad libitum.
Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

50%, 25% and 10%. 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear

No. of animals per dose:

4

Details on study design:

Groups of four mice were treated with the test item undiluted diluted at 50%, 25% and 10% in N,N-dimethylformamide (DMF). The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). The test item 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 four mice received the vehicle alone in the same manner.

Positive control substance(s):

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

Results and discussion

Positive control results:

An EC1.6 value of 14.41% was observed for the test item α-Hexylcinnamaldehyde, resulting in a classification as category 1 “Skin sensitisation” in accordance with the Regulation (EC) No. 1272/2008.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.01

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

1.05

Test group / Remarks:

25%

Results 3

Key result

Parameter:

SI

Value:

1.36

Test group / Remarks:

50%

Any other information on results incl. tables

Clinical observations and mortality

No mortality and no signs of systemic toxicity were noted in the test and control animals during the

test.

Weight evolution

No statistical significant change in body weight was noted for all treated groups versus control group

(Student’s test).

Estimation of the proliferative response of lymph node cells

No stimulation index higher than 1.6 was recorded whatever the tested concentrations.

The Stimulation Index (SI) calculated by individual approach was 1.01, 1.05 and 1.36 for the treated groups at 10%, 25% and 50%, respectively.

Therefore, the EC1.6 cannot be determined due to the absence of SI value higher than 1.6.

Local irritation

Residual test item was noted in all animals treated at 50% between Days 2 and 6.

Red coloration not preventing quotation of erythema was noted in animals treated at 10% between Days 3 and 5 and in animals treated at 25% between Days 3 and 6.

No increase in ear thickness and in ear weight was noted in animals treated at 10%, 25% and 50%, respectively.

Therefore, the test item has to be considered as not excessively irritant at these concentrations.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained, under these experimental conditions, enable to conclude that the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) does not have to be classified as a skin sensitizer, in accordance with the Regulation EC No. No. 1272/2008 on classification, labelling and packaging of substances and mixtures.
No signal word or hazard statement is required.

Executive summary:

The test was performed to assess the skin sensitisation potential of the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) in the CBA/J strain mouse following topical applications to the dorsal surface of the ear. The basic principle underlying the LLNA:BrdU is that sensitizers induce proliferation of lymphocytes in the lymph nodes draining the site of test item application.

Three groups of four animals were treated for three consecutive days (D1, D2, D3) with 50 μL (25 μL per ear) of the test item diluted at concentrations of 10%, 25% and 50% in N,N-dimethylformamide (DMF)

A further group of four animals was treated with DMF.

On D5, 0.5 mL of BrdU solution (10 mg/mL) was injected by the intraperitoneal route.

On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte using an ELISA kit.

The experimental protocol was established according to the O.E.C.D. Guideline No. 442-B adopted 25 June 2018.

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.

Residual test item was noted in all animals treated at 50% between Days 2 and 6.

Red coloration not preventing quotation of erythema was noted in animals treated at 10% between Days 3 and 5 and in animals treated at 25% between Days 3 and 6.

No increase in ear thickness and in ear weight was noted in animals treated at 10%, 25% and 50%, respectively.

Therefore, the test item has to be considered as not excessively irritant at these concentrations.

The Stimulation Index (SI) calculated by individual approach was 1.01, 1.05 and 1.36 for the treated

groups at 10%, 25% and 50%, respectively.

Therefore, the EC1.6 cannot be determined due to the absence of SI value higher than 1.6.

The results obtained, under these experimental conditions, enable to conclude that the test item Acetic acid, oxo-, sodium salt, reaction products with ethanolamine and phenol, sodium hydroxide and ferric chloride (MEAHA Fe) does not have to be classified as a skin sensitizer, in accordance with the Regulation EC No. No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

No signal word or hazard statement is required.