Methyl hydrogen fumarate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2021- July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

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:

Methyl hydrogen fumarate

EC Number:

220-412-6

EC Name:

Methyl hydrogen fumarate

Cas Number:

2756-87-8

Molecular formula:

C5H6O4

IUPAC Name:

4-methoxy-4-oxobut-2-enoic acid

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Remarks:

Inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: [yes]
- Microbiological status of animals, when known:
- Age at study initiation: Young adult animals (approximately 10 weeks old) were selected.
- Weight at study initiation: 23.1 to 28.2 g
- Housing: On arrival and following assignment to the study, animals were group housed (up to 5
animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon
MIII type; height 18 cm.) containing sterilized wooden fibers as bedding material (Lignocel S
8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with
water bottles. The rooms in which the animals were kept were documented in the study
records.
Animals were separated during designated procedures/activities. Each cage was clearly
labeled.
- Diet (e.g. ad libitum): Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was
provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental
contaminants. Results of the analysis were provided by the supplier and are on file at the Test
Facility.
It is considered that there were no known contaminants in the feed that would interfere with
the objectives of the study.
- Water (e.g. ad libitum): Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the
Test Facility.
It is considered that there were no known contaminants in the water that would interfere with
the objectives of the study.
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at
least 5 days before the commencement of dosing.
- Indication of any skin lesions: Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from
the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C) & Humidity (%): Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were
maintained. The actual daily mean temperature during the study period was 22 to 23°C with
an actual daily mean relative humidity of 44 to 67%.
- Air changes (per hr): Ten or greater air changes per hour with 100% fresh air (no air recirculation)
were maintained in the animal rooms.
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle was maintained.
- IN-LIFE DATES: From: 21 May 2021 To: 14 June 2021

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Remarks:

The vehicle was chosen from the vehicles specified in the test guideline (in order of preference): Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol, dimethylsulfoxide and 1% Pluronic© L92 in Elix water

Concentration:

2, 5 and 10% w/w

No. of animals per dose:

5 (five)

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The vehicle was selected on the basis of maximizing the solubility based
on trial preparations performed at Charles River Den Bosch and on information provided by
the Sponsor. There was no information available about the stability and solubility of the test item
in vehicle.
A pre-screen test was conducted in order to select the highest test item concentration to be
used in the main study. In principle, this highest concentration should cause no systemic
toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2
and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that
can technically be applied.
Two test item concentrations were tested; a 10% and 20% concentration. The highest
concentration was the highest concentration that could be prepared homogeneously.
The test system, procedures and techniques were identical to those used in the main study
except that the assessment of lymph node proliferation and necropsy were not performed.
Two young adult females per concentration were selected. Each animal was treated with one
concentration on three consecutive days. Animals were group housed in labeled Makrolon
cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital
thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
Animals were assigned to the study at the discretion of the coordinating biotechnician, with
all animals within ± 20% of the sex mean body weights. Animals in poor health or at
extremes of body weight range were not assigned to the study.

Three groups of five animals were treated with one test item concentration per group. The
highest test item concentration was selected from the pre-screen test. One group of five
animals was treated with the vehicle.


- Criteria used to consider a positive response:
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI)
is calculated for each group using the individual SI values. The individual SI is the ratio of the
DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI = 3, the test item may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
Preparation of Test Item
The test item was stored in the refrigerator and was allowed to warm to room temperature for
at least 30 minutes before use. Test item dosing formulations (w/w) were homogenized to
visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the
vehicle to the test item.
The dosing formulations were kept at room temperature until dosing. The dosing formulations
were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and no correction was made for
the purity/composition of the test item, since the test method requires a logical concentration
range rather than specific dose levels.
Any residual volumes were discarded.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 µL/ear) with the test item, at
approximately the same time on each day. The formulations were stirred with a magnetic
stirrer until dosing.
The control animals were treated in the same way as the experimental animals, except that the
vehicle was administered instead of the test item.

Positive control substance(s):

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

Results and discussion

Positive control results:

The SI values calculated for the item concentrations 5, 10 and 25% were 1.2, 2.2 and 5.6,
respectively. An EC3 value of 13.5% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The
results of the 6 monthly HCA reliability checks of the recent years were 16.3, 12.8, 9.0, 10.9
and 8.0%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at
Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
The raw data, study plan and report from this study are kept in the Charles River Den Bosch
archives. The test described above was performed in accordance with Charles River Den
Bosch Standard Operating Procedures and the report was audited by the QA-unit.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1

Variability:

SEM= 0.2 Median SI = 1

Test group / Remarks:

Control

Results 2

Parameter:

SI

Value:

2.4

Variability:

SEM = 0.8 Median SI = 2.1

Test group / Remarks:

2% w/w

Results 3

Parameter:

SI

Value:

2.1

Variability:

SEM = 0.8 Median SI = 1.6

Test group / Remarks:

5% w/w

Results 4

Parameter:

SI

Value:

1.8

Variability:

SEM = 0.4 Median SI= 2.2

Test group / Remarks:

10% w/w

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA - See table 1
Since the within group variation was high, both mean and median values were used to
interpret the data.
Mean DPM/animal values for the experimental groups treated with test item concentrations 2,
5 and 10% were 950, 828 and 721 DPM, respectively. The mean DPM/animal value for the
vehicle control group was 390 DPM. The SI values calculated for the test item concentrations
2, 5 and 10% were 2.4, 2.1 and 1.8, respectively.
Median DPM/animal values for the experimental groups treated with test item concentrations
2, 5 and 10% were 794, 595 and 800 DPM, respectively. The median DPM/animal value for
the vehicle control group was 372 DPM. The SI values calculated for the test item
concentrations 2, 5 and 10% were 2.1, 1.6 and 2.2, respectively.
DETAILS ON STIMULATION INDEX CALCULATION
All results presented in the tables of the report are calculated using values as per the raw data
rounding procedure and may not be exactly reproduced from the individual data presented.
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI)
is calculated for each group using the individual SI values. The individual SI is the ratio of the
DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI = 3, the test item may be regarded as a skin sensitizer.
EC3 CALCULATION
Not applicable
CLINICAL OBSERVATIONS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals.
BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as
controls over the study period.
SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).
Body weights and body weight gain of experimental animals remained in the same range as
controls over the study period. No irritation was observed in any of the animals.

Any other information on results incl. tables

Prescreen test: At 20%, variation in ear thickness during the observation period were more than 25% from
Day 1 pre-dose values and therefore this concentration did not meet the selection criteria.
At 10%, no signs of systemic toxicity were noted and only very slight irritation was observed.
Therefore, this concentration was selected as highest concentration for the main study.

Table 1 : Main Study: Relative Size Lymph Nodes, Radioactivity Counts (DPM) and
Stimulation Index (SI)

Group	TS (%w/w)	Animal	Size nodes		DPM/animal	Mean DPM ± SEM		Mean SI ± SEM		Median DPM	Median SI
			Left	right
1	0	1	n	n	372	390	63	1.0	0.2	372	1.0
		2	n	n	302
		3	n	n	400
		4	n	n	254
		5	n	n	622
2	2	6	n	n	608	950	327	2.4	0.8	794	2.1
		7	n	n	2132
		8	n	n	794
		9	n	n	1037
		10	n	n	178
3	5	11	n	n	595	828	312	2.1	0.8	595	1.6
		12	n	n	1476
		13	n	n	258
		14	n	n	1659
		15	n	n	154
4	10	16	n	n	800	721	161	1.8	0.4	800	2.2
		17	n	n	578
		18	n	n	171
		19	n	n	1070
		20	n	n	985

TS = test item (% w/w).
Relative size auricular lymph nodes (-, -- or ---: degree of reduction, +,++ or +++: degree of enlargement, n:
considered to be normal).
DPM = Disintegrations per minute.
SEM = Standard Error of the Mean.

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since both the median and mean SI values were all below the threshold value of SI = 3, no
abnormalities of the lymph nodes were observed and because no clear dose-response was
present, there was no indication that Monomethyl Fumarate (MMF) could elicit a SI = 3 when
tested up to 10%. It was established that the EC3 value (the estimated test item concentration
that will give a SI =3) (if any) exceeds 10%.
The six-month reliability check with Alpha-Hexylcinnamaldehyde indicates that the Local
Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for
testing for contact hypersensitivity (see Appendix 4).
Based on these results, Monomethyl Fumarate (MMF) would not be regarded as a skin
sensitizer according to the recommendations made in the test guidelines. The test item does
not have to be classified and has no obligatory labelling requirement for sensitization by skin
contact according to the Globally Harmonized System of Classification and Labelling of
Chemicals (GHS) of the United Nations (2017) (including all amendments) and the
Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and
mixtures (including all amendments).

Executive summary:

The objective of this study was to evaluate whether Monomethyl Fumarate (MMF) induces
skin sensitization in mice after three epidermal exposures of the animals under the conditions
described in this report.
The study was carried out based on the guidelines described in:
• OECD, Section 4, Health Effects, No.429 (2010).
• EC No 640/2012, Part B: "Skin Sensitization: Local Lymph Node Assay".
• EPA, OPPTS 870.2600 (2003) “Skin Sensitization”.
The six-month reliability check with Alpha-Hexylcinnamaldehyde indicates that the Local
Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for
testing for contact hypersensitivity.
Test item concentrations selected for the main study were based on the results of a pre-screen
test. At 20%, variation in ear thickness during the observation period were more than 25%
from Day 1 pre-dose values and therefore this concentration did not meet the selection
criteria. At 10%, no signs of systemic toxicity were noted and only very slight irritation was
observed. Therefore, 10% was selected as highest concentration for the main study.
In the main study, three experimental groups of five female CBA/J mice were treated with
test item concentrations of 2, 5 or 10% w/w on three consecutive days, by open application on
the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (N,Ndimethylformamide
(DMF)). Three days after the last exposure, all animals were injected
with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were
excised and pooled for each animal. After precipitating the DNA of the lymph node cells,
radioactivity measurements were performed. The activity was expressed as the number of
disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated
for each group.
All auricular lymph nodes of the animals of the experimental and control groups were
considered normal in size. No macroscopic abnormalities of the surrounding area were noted
for any of the animals.
Since the within group variation was high, both mean and median values were used to
interpret the data. Mean DPM/animal values for the experimental groups treated with test
item concentrations 2, 5 and 10% were 950, 828 and 721 DPM, respectively. The mean
DPM/animal value for the vehicle control group was 390 DPM. The SI values calculated for
the test item concentrations 2, 5 and 10% were 2.4, 2.1 and 1.8, respectively. Median
DPM/animal values for the experimental groups treated with test item concentrations 2, 5 and
10% were 794, 595 and 800 DPM, respectively. The median DPM/animal value for the
vehicle control group was 372 DPM. The SI values calculated for the test item concentrations
2, 5 and 10% were 2.1, 1.6 and 2.2, respectively.
Since both the median and mean SI values were all below the threshold value of SI = 3, no
abnormalities of the lymph nodes were observed and because no clear dose-response was
present, there was no indication that Monomethyl Fumarate (MMF) could elicit a SI ≥ 3 when
tested up to 10%. It was established that the EC3 value (the estimated test item concentration
that will give a SI =3) (if any) exceeds 10%.

Based on these results, Monomethyl Fumarate (MMF) would not be regarded as a skin
sensitizer according to the recommendations made in the test guidelines. The test item does
not have to be classified and has no obligatory labelling requirement for sensitization by skin
contact according to the Globally Harmonized System of Classification and Labelling of
Chemicals (GHS) of the United Nations (2017) (including all amendments) and the
Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and
mixtures (including all amendments).