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Toxicological information

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:
November 07-30, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP lab following OECD guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Glycerol formal
IUPAC Name:
Glycerol formal
Test material form:
other: liquid
Details on test material:
Name : Glycerol formal
Other name : Glycerol Formal Ultra Pure Grade
Both denominations correspond to the same test item.
CAS number : 4740-78-7 and 5464-28-8 (~ 50/50 ratio)
Batch number : 1206191600R
Description : colourless liquid
Container : brown glass flask
Purity : 99.9840%
Storage condition : at room temperature
Date of receipt : 12 July 2012
Expiry date : 19 June 2015.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Animals

Number:
preliminary test: 4 nulliparous and non pregnant females,
main test: 28 nulliparous and non pregnant females.

Species and sanitary status: CBA/J mice.

Reason for selection of species: this inbred strain was chosen on the basis of previous studies performed in our laboratory, in which CBA/J mice showed the best proliferative response. Females have been chosen since this sex is recommended by Regulatory Authorities for this type of study.

Breeder: Janvier, Le Genest-Saint-Isle, France.

Age/weight: on the beginning of the treatment period, the animals of the preliminary test were approximately 10 weeks old and had a mean body weight of 21.8 g (range: 20.3 g to 23.2 g) and the animals of the main test were approximately 8 weeks old and had a mean body weight of 20.2 g (range: 18.3 g to 22 g).

Receipt: on arrival, the animals were given a clinical examination to ensure that they are in good condition.

Allocation to groups: on arrival, the animals were allocated to the groups using a manual randomization procedure. A larger number of animals than necessary were acclimated to permit the selection and/or replacement of individuals.

Acclimation: the animals were acclimated to the study conditions for a period of six days before the beginning of the treatment period. At the end of acclimation period, the required number of animals was selected according to clinical condition and body weight.

Identification: the animals were individually identified on the tail using an indelible marker (unique CiToxLAB France identity number).

Environmental conditions
From arrival at CiToxLAB France, the animals were housed in a barriered rodent unit.
The animal room conditions were set as follows:
 temperature: 22 ± 2°C,
 relative humidity: 50 ± 20%,
 light/dark cycle: 12 h/12 h,
 ventilation: approximately 12 cycles/hour of filtered, non-recycled air.

The corresponding instrumentation and equipment are checked and calibrated at regular intervals. The temperature and relative humidity are recorded continuously and the records checked daily and filed.

Housing
The animals were housed by group of two (preliminary test) or four (main test) in polycarbonate cages (Tecniplast 1145T, 435 cm2) containing autoclaved sawdust (SICSA, Alfortville, France).
Each cage contained two enrichments (mouse hut and cocoon).
In the main test, on day 6 before the 3H-TdR injections, the animals were individually housed in disposable crystal polystyrene cages (22.0 cm x 8.5 cm x 8.0 cm).

Food and water
All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch Nos. 2827489 and 2537604, (SSNIFF Spezialdiäten GmbH, Soest, Germany) and tap water (filtered using a 0.22 micron filter) contained in bottles.

Contaminant analyses
The batches of diet and sawdust were analyzed by the suppliers for composition and contaminant levels.
Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants that could have interfered with or prejudiced the outcome of the study were found in the diet, drinking water or sawdust.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
5, 10, 25, 50 or 100%
No. of animals per dose:
4
Details on study design:
TREATMENT

A) Rationale for concentration selection
The maximum concentration tested in the main test was selected according to the criteria specified in the OECD Guidelines and on the basis of the data that was obtained in the preliminary test:
 the vehicle should be selected on the basis of producing a homogeneous preparation suitable for application of the test item,
 the concentrations were selected from the concentration series 100% (when test item can be sampled by a pipette), 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc,
 the maximum concentration of the test item was selected to avoid overt systemic toxicity and excessive local skin irritation the latter being defined by an > 25% increase of the ear thickness.

B) Preliminary test

B.1) Treatment groups
To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was performed in 4 animals.

The treatment groups for the preliminary test are detailed in the following table:

Group Number of animals Concentration (%)
Left ear Right ear
1 2 females 10 25
2 2 females 50 100

B.2) Application
On days 1, 2 and 3, at approximately the same time each day, a dose-volume of 25 µL of the appropriate dose formulation preparation was applied to the dorsal surface of both ears (one concentration per ear), using an adjustable pipette fitted with a plastic tip.
In order to avoid licking, to ensure an optimized application of the test materials and to facilitate ear thickness measurement, the animals were placed under light isoflurane anesthesia during the administration.
No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.

C) Main test

C.1) Treatment groups
The treatment groups for the main test are detailed in the following table:

Group Number of animals Treatment Concentration (%)
3 4 females Vehicle 0
4 4 females Test item 5
5 4 females Test item 10
6 4 females Test item 25
7 4 females Test item 50
8 4 females Test item 100
9 4 females Positive control item 25% of HCA

C.2) Application
On days 1, 2 and 3, at approximately the same time each day, a dose-volume of 25 µL of the control or dose formulation preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
No massage was performed but the tip was used to spread the preparation over the application site. No rinsing was performed.

CLINICAL EXAMINATIONS
A) Morbidity and mortality
Each animal was checked for mortality and morbidity once a day during the acclimation, treatment and observation periods, including weekends and public holidays.

B) Clinical signs
Each animal was observed once a day for the recording of clinical signs.

C) Body weight
The body weight of each animal was recorded once during the acclimation period, and then on the first day of treatment and on the day of sacrifice.

D) Ear thickness measurements and recording of local reactions
Ear thickness measurements and recording of local reactions were performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses.

On days 1, 2 and 3 (before each cutaneous application) and on day 6 (immediately after sacrifice), the thickness of both ears of each preliminary test animal (groups 1 and 2) and the left ear of each main test animal (groups 3 to 8) was measured, using a micrometer.
No measurement of ear thickness was performed for the main test animals of the positive control group (group 9).

The irritation level of the test item was determined according to the following table:

% increase in ear thickness
between day 1 and day 6 Irritation level Interpretation
< 10% I Non-irritant
≥ 10 – < 25% II Slightly irritant
≥ 25% III Irritant

AURICULAR LYMPH NODE CELL (ALNC) PROLIFERATION ASSAY

A) Intravenous injection of 3H-TdR and sampling of auricular lymph nodes
Lymph node cell proliferative response was measured as described by Kimber and Dearman (1991). On day 6 of the main test, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 µCi of 3H-TdR (specific activity of 20 Ci/mmol) via the tail vein.
Approximately 5 hours later, the main test animals were sacrificed by an intraperitoneal injection of pentobarbital sodium followed by a cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group. A single cell suspension of ALNC was prepared by mechanical disaggregation in Petri dishes with the plunger of a syringe.

B) Preparation of auricular lymph node cell suspensions and determination of proliferation
Cell suspensions were washed once with 15 mL of 0.9% NaCl. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic (TCA) at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three milliliters of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using B-scintillation counting.
The results were expressed as disintegrations per minute (dpm) per group and as dpm/node.

Stimulation Indices (SI) were calculated according to the following formula:

dpm per node of the treated group
SI = --------------------------------------
dpm per node of the vehicle control group

C) Acceptance criteria
The study is considered valid if the SI for the positive control is higher than the threshold positive value of 3.

D) Evaluation of results
The test item is considered as a skin sensitizer when the SI for a dose group is ≥ 3 together with consideration of a dose-response relationship. Other relevant criteria such as radioactivity levels and ear thickness are also taken into account to evaluate the data.

E) Classification
The test item was classified according to the criteria of Commission Regulation (EU) No. 286/2011 of 10 March 2011 amending for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No. 1272/2008 (CLP) of the European parliament and of the council of 16 December 2008 on classification, labeling and packaging of substances and mixtures.

A test item being positive in the LLNA was classified Category 1 and sub-categorized as follows:
 sub-category 1A if EC3 value ≤ 2%,
 sub-category 1B if EC3 value > 2%.

In addition, it was attributed the following information:
 hazard statement: H317: may cause an allergic skin reaction,
 signal word: "warning".


PATHOLOGY: fate of the animals
On completion of the observation period, all preliminary test animals were sacrificed by an intraperitoneal injection of pentobarbital sodium followed by a cervical dislocation.
Approximately 5 hours after intravenous injection of 3H-TdR, all surviving main test animals were sacrificed by an intraperitoneal injection of pentobarbital sodium followed by a cervical dislocation.

Any local reaction was also recorded (coloration of the skin, erythema, scabs…).

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group (SI = 10.73). The experiment was therefore considered valid.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.17
Test group / Remarks:
100%
Key result
Parameter:
SI
Value:
1.41
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
1.15
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
1.27
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
1.65
Test group / Remarks:
5%
Parameter:
SI
Remarks on result:
other: 1.65 for GF tested at 5% 1.27 for GF tested at 10% 1.15 for GF tested at 25% 1.41 for GF tested at 50% 1.17 for GF tested at 100% No notable lymphoproliferation was noted with the test item at any tested concentrations.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Please refer to Table 1 under "Any other information on results incl. tables"

Any other information on results incl. tables

Table 1 - Proliferation assay (mean values)

 

Groups

Treatment and conc.

Number of nodes per group 

dpm per group 

dpm per node 

SI

Increase in earthickness (% between D1 and D6)

Irritation level

3

Vehicle

8

1127

140.88

-

-3.00

I

4

GF 5%

8

1857

232.13

1.65

1.03

I

5

GF 10%

8

1076

179.33

1.27

-1.35

I

6

GF 25%

8

1298

162.25

1.15

-1.05

I

7

GF 50%

8

1585

198.13

1.41

0.00

I

8

GF 100%

8

1314

164.25

1.17

-2.02

I

9

HCA 25%

8

12098

1512.25

10.73

-

-

 

I : non-irritant (increase in ear thickness <10%)

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions of this study, the test item, Glycerol formal, gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.

Therefore, the test item should not be classified as a skin sensitizer according to the criteria of CLP Regulation.
Executive summary:

The objective of this study was to evaluate the potential of the test item, Glycerol formal, to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA).

 

Methods

 

To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test item, Glycerol formal, by topical route to the dorsal surface of both ears (one concentration per ear) on days 1, 2 and 3 at concentrations of 10, 25, 50 or 100% under a dose‑volume of 25 µL.From day 1 to day 3 then on day 6, the thicknessof both ears of each animal was measured and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recordedonce during the acclimation period, and thenon days 1 and 6.On completion of the observation period, the animals were sacrificedthen discarded without macroscopic post-mortem examination.

 

In the main test, five groups of four female mice received the test item by topical routeto the dorsal surface of both earson days 1, 2 and 3at concentrations of 5, 10, 25, 50 or 100% under a dose‑volume of 25 µL. One negative control group of four females received the vehicle (dimethylformamide (DMF)) under the same experimental conditions. Additionally, one positive control group of four females received the positive control, α‑hexylcinnamaldehyde (HCA), at 25%in a mixture Acetone/Olive Oil (4/1; v/v)under the same experimental conditions.

From day 1 to day 3 then on day 6, the thicknessof the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recordedonce during the acclimation period, and thenon days 1 and 6.

After 2 days of resting, on day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of3H-TdR.

The results were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

 

Results

 

Mortality and clinical signs

Animal 50 given the test item at 10% was found dead on day 6. No other unscheduled deaths occurred during the main test.

No clinical signs indicative of systemic toxicity were observed in any animals.

 

Local irritation

No local reactions and no notable increase in ear thickness were observed in any tested concentrations.

 

Proliferation assay

The SI of the positive control was > 3; this experiment was therefore considered valid.

No notable lymphoproliferation was noted with the test item at any tested concentrations.

 


Conclusion

 

Under the experimental conditions of this study, the test item, Glycerol formal, gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties.

 

Therefore, the test item should not be classified as a skin sensitizer according to the criteria of CLP Regulation.