Glycerol monomyristate

Endpoint summary

Description of key information

Skin sensitisation: not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

12 Nov 1984 - 18 Apr 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Lack of test material details, no reliability check.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

lack of test materials details, no reliability check.

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

yes

Remarks:

lack of test materials details, no reliability check.

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was performed prior to the amendment of Regulation (EC) No. 1907/2006 requiring the Local Lymph Node Assay to be performed as the first-choice method for in vivo testing.

Species:

guinea pig

Strain:

other: Pirbright white

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Weight at study initiation: 312 g (test group), 303 g (control group)
- Housing: 5 animals per cage in Makrolon Type IV
- Diet: Altromin Diet 3032 DK (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50-60
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal and epicutaneous

Vehicle:

other: paraffin oil (1%) for intradermal application and vaseline (50%) for epicutaneous application

Concentration / amount:

Induction:
Intradermal: 1 %
Epicutaneous: 50%

Challenge: 25%

Route:

epicutaneous, occlusive

Vehicle:

other: paraffin oil (1%) for intradermal application and vaseline (50%) for epicutaneous application

Concentration / amount:

Induction:
Intradermal: 1 %
Epicutaneous: 50%

Challenge: 25%

No. of animals per dose:

20

Details on study design:

RANGE FINDING TESTS:
3 preliminary tests were performed with 5 animals each:
Intradermal application with a concentration of 1% of test substance in vaseline induced skin irritation (definite effects, without induction of necrosis). After epidermal application with a concentration of 50% of the test substance in vaseline, this concentration was determined as the minimal irritating concentration. To be able to determine the maximum non-irritating concentration for challenge treatment, intracutaneous injections of 0.1 mL FCA bilaterally of the vertebral column were made. 2 weeks later 25% and 50% test substance concentrations were applied for 24 h under plaster-covering. A concentration of 25% was found to be the maximum non-irritating concentration.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections):
Injection 1: FCA
Injection 2: test substance in paraffin
Injection 3: test substance in a 1:1 mixture (v/v) FCA
Epicutaneous: test substance in vaseline

- Control group:
Injection 1: FCA
Injection 2: paraffin
Injection 3: parafin in a 1:1 mixture (w/v) FCA
Epicutaneous: vaseline

- Site: shoulder region (intradermal + epicutanous)
- Frequency of applications: every 7 days
- Duration: Days 0-8
- Concentrations: intradermal 1%, epicutaneous 50%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 20
- Exposure period: 24 h
- Test groups: test substance and vehicle only
- Control group: test substance and vehicle only
- Site: cranial (vehicle) and caudal (test substance)
- Concentrations: 25%
- Evaluation (hr after challenge): 48 and 72

Positive control substance(s):

not specified

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

induction: 0%; challenge: 25%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: induction: 0%; challenge: 25%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

induction: 1%; challenge: 25%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: induction: 1%; challenge: 25%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

induction: 0%; challenge: 25%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: induction: 0%; challenge: 25%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

induction: 1%; challenge: 25%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: induction: 1%; challenge: 25%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

1st reading

Group:

positive control

Remarks on result:

other: no positive control group included

No effects on bodyweight were observed during the study period. After intradermal injection all animals showed the expected reactions for FCA. Intradermal injections of the vehicle paraffin caused reactions as well (no further information). After removal of the epicutaneous induction patch erythemateous reaction was observed being clearly decreased 24 h later. According to the author, individual results were not given in tabulated form, as all skin examinations were negative, both at 24 and 48 h after challenge exposure.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

CLP: not classified
DSD: not classified

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 Nov - 22 Nov 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Modified Local Lymph Node Assay (Integrated Model for the Differentiation of Skin Reactions, IMDS) accordimng to Vohr, H.-W. et al. (Arch Toxicol 73:501-509, 2000) and Ehling, G. et al. (Toxicology 212: 60-68 and 89-79, 2005).

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Modified Local Lymph Node Assay (Integrated Model for the Differentiation of Skin Reactions, IMDS) accordimng to Vohr, H.-W. et al. (Arch Toxicol 73:501-509, 2000) and Ehling, G. et al. (Toxicology 212: 60-68 and 89-79, 2005).

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: Hsd Win:NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 9 weeks
- Weight at study initiation: 23-29 g
- Housing: during the adaptation period, up to 8 mice were housed together in conventional Makrolon type III cages, while during the study period the animals were single-housed in type II cages. During adaptation period, the cages were changed at least twice a week. Low-dust wood shavings were used as bedding.
- Diet: PROVIMI KLIBA SA 3883 maintenance diet for rats and mice (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40-70
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

2, 10 and 50%

No. of animals per dose:

6

Details on study design:

RANGE FINDING TESTS:
A range finding test was not conducted. According to the author, based on in-house experience with the test system and the available information on the properties of the test item, the following concentrations were used: 0 (vehicle control), 2, 10 and 50%."

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node weight and cell count determinations
- Criteria used to consider a positive response:
Cell count index: The "positive level" for the cell count index was stated to be 1.4.
Ear swelling: The "positive level" of ear swelling was stated to be a 2E-02 mm increase (about 10% of the control values)
The author explicitly stated that the cut-off values for "positive levels" mentioned above are exclusively defined for the NMRI outbred mice used for this study (Homey, B. et al. [1998] Toxicol. and Appl. Pharmacol. 153:83-94; Vohr, H.-W. et al. [2000] Arch. Toxicol. 73:501-509). Further, the author noted that such positive limits have to be calculated for each strain of mice individually (Ehling, G. et al. [2005] Toxicology 212:60-68; Ehling, G. et al. [2005] Toxicology 212:69-79).

TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation or the vehicle was applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25 µL/ear.

INVESTIGATIONS

- Autopsies:
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (Day 4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
- LLN Weight and cell count determinations:
The weight of the lymph nodes was determined on a semiautomatic balance and stored electronically. After crushing the lymph nodes through a sieve in a 12-well plate and dispersion in 2 mL PBS, the cell counts per mL were determined using an electronic cell counter. These data were also directly collected and processed by a computer. Mean values, indices and standard deviations were calculated by a spreadsheet software.
A special software was used to calculate mean values and standard deviations of lymph node weights. Indices were calculated manually.
The so-called stimulation (or LLN-) index is calculated by dividing the absolute number of weight or cell counts of the substance-treated lymph nodes by the vehicle-treated ones. Thus, in case of no stimulating effect, the index is always about 1.00 (± standard deviation), and the indices of vehicle-treated animals are set to 1.00 (± standard deviation).
- Ear swelling:
Before the first treatment and before sacrifice, the thickness of both auricles of the animals was measured using a spring-loaded micrometer. Mean values, indices and standard deviations of the ear swelling were calculated by a spreadsheet software.
- Ear weight:
On Day 4 of the study, the ear weight of the sacrificed animals was measured using a punch to take a piece of every ear with a diameter of 8 mm. The weights were determined on a semiautomatic balance. Mean values, indices and standard deviations of the ear weights were calculated by a spreadsheet software.
- Body weight:
The body weights of the animals were recorded at the start and at the end of the study (Day 1 and Day 4).

Positive control substance(s):

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

Statistics:

When it was statistically reasonable, the values from the treated groups were compared with those from the control group(s; vehicle) by a one-way analysis of variance (ANOVA) when the variances were considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances were considered heterogeneous (p ≤ 0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5%. Two-sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99% by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffe'S method, which can be used for both equal and unequal sample sizes.
In this method of statistical processing of measurements a large number of comparisons is made, and as a result of the multiple tests the overall probability of error is considerably greater than the p values suggest (increased number of false-positive results). On the other hand, the known methods of adjusting p values lead to an excessive increase in the number of false negatives. In view of these problems, the biological and toxicological relevance was also taken into consideration in the evaluation of statistical significance.
For this reason, in the case of indices only the standard deviations between groups and difference analysis of the mean values were used in the evaluation of the biological relevance.

Key result

Parameter:

SI

Value:

0.84

Test group / Remarks:

2%

Key result

Parameter:

SI

Value:

0.95

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

0.97

Test group / Remarks:

10%

Key result

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see remarks

Remarks:

No significant changes were observed in the absolute number of lymph node cell counts per mL (see Table 2 for details) Group mean cell counts (1000 cells/mL lymph node suspension ± standard deviation): Vehicle control: 10961,50 ± 3006.68 2%: 9169.42 ± 3009.43 10%: 10421.25 ± 2096.75 50%: 10629.50 ± 2149.53 No significant changes were observed in ear thickness between Day 1 and Day 4 as well as between animals treated with the test item and with the vehicle (see Table 3 for details). Group mean ear swelling (Day 1 / Day 4; in 0.01 mm ± standard deviation in %): Vehicle control: 17.83 ± 3.24 / 17.25 ± 2.62 2%: 17.67 ± 3.69 / 17.92 ± 2.87 10%: 17.08 ± 3.01 / 17.17 ± 4.86 50% 17.33 ± 5.12 / 17.67 ± 4.41

Table 2. Individual cell counts

		Cell count (1000 cells/mL lymph node suspension)
Treatment group	Animal No.	Arithmetic mean (n = 2)	Group mean	SD	SD (%)	Cell count index
Vehicle	1	10541.5	10961.50	3006.68	27.43	1.00
	2	13500.5
	3	11383
	4	11254.5
	5	13686
	6	5403.5
2%	7	7443	9169.42	3009.43	32.82	0.84
	8	9853.5
	9	9090.5
	10	6253.5
	11	14736
	12	7640
10%	13	10796.5	10421.25	2096.75	20.12	0.95
	14	9343
	15	7629.5
	16	14002
	17	10206.5
	18	10550
50%	19	7714.5	10629.50	2149.53	20.22	0.97
	20	8873.5
	21	12331.5
	22	11729.5
	23	13247.5
	24	9880.5

Table 3. Ear swelling (6 animals per group, in 0.01 mm).

Dose (%)	Day 1 (mean ± SD in %)	Day 4 (mean ± SD in %)	Index Day 4
0 (vehicle)	17.83 ± 3.24	17.25 ± 2.62	1.00
2	17.67 ± 3.69	17.92 ± 2.87	1.04
10	17.08 ± 3.01	17.17 ± 4.86	1.00
50	17.33 ± 5.12	17.67 ± 4.41	1.02

Table 4. Ear and lymph node weight.

	Ear weight (6 animals per group, in mg per 8 mm punch)		Lymph node weight (6 animals per group)
Dose (%)	Day 4 (mean ± SD in %)	Index Day 4	Weight index (index of mean ± SD in %)
0 (vehicle)	11.44 ± 6.52	1.00	1.00 ± 20.70
2	11.28 ± 4.64	0.99	0.99 ± 28.19
10	11.43 ± 9.14	1.00	1.00 ± 23.44
50	12.18 ± 7.94	1.06	1.02 ± 20.02

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.

Conclusions:

CLP: not classified
DSD: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Justification for read-across

Data on the skin sensitisation potential of Glycerol monomyristate (CAS 27214 -38 -6) are not available. The assessment of skin sensitisation was therefore based on data from source substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Animal data

The skin sensitising potential of C12: Glycerides, C8-21 and C8-21-unsatd., mono- and di-, acetates (CAS 97593 -30 -1) was investigated in a modified Local Lymph Node Assay (LLNA) in mice according to OECD guideline 429 and in compliance with GLP (Lanxess, 2008). In this study, 6 female Hsd Win:NMRI mice per test group were treated with test substance at concentrations of 2, 10 and 50% in acetone/olive oil (4:1 v/v) or with the vehicle alone. The test substance formulations or the vehicle were applied epicutaneously onto the dorsal part of each ear (25 µL/ear) for three consecutive days. In order to assess skin irritation, the thickness of both auricles of the animals was measured before the first treatment and prior to sacrifice. In addition, ear weights were determined at necropsy. On Day 4, animals were sacrificed and weight of the lymph nodes was determined. The cell proliferation of pooled lymph nodes from individual animals was measured by counting the cells in suspension using an electronic cell counter. The mean cell count (1000 cells/mL lymph node suspension) for each test group was 9169, 10421 and 10630 at concentrations of 2, 10 and 50% of the test substance, respectively. Treatment with the test substance did not result in a significant increase of absolute number of lymph node cell counts per mL compared to control (cell count = 10962). Based on these results, cell count indices of 0.84, 0.95 and 0.97 were calculated for treatment concentrations of 2, 10 and 50%, respectively. A positive result in this strain of mice was obtained if the cell count index was ≥ 1.4. No local or systemic toxicity and no effects on body weights were observed. No effects on ear weights and ear swelling were observed in the treated animals compared to controls. The historical positive control alpha hexyl cinnamic aldehyde at concentrations of 3, 10 and 30% confirmed the sensitivity and reliability of the experimental technique. Under the above mentioned conditions, the test substance was not found to be a sensitiser in the modified LLNA.

 

The skin sensitising potential of Glycerides, C16-18 and C18-hydroxy mono- and di- (CAS 91845 -19 -1) was studied in guinea pigs according to the maximisation method (OECD guideline 406) and in compliance with GLP (BASF, 1985). In three preliminary tests, suitable treatment concentrations for the main study were determined in each 5 animals. For the intradermal route, the test substance at concentrations of 1 and 10 % in paraffin was administered to the clipped skin of 5 animals. Based on the induction of moderate irritant effects on the skin but no necrosis, a test substance concentration of 1% in paraffin oil (w/w) was chosen for intradermal injections in the main study. For the cutaneous route, the test substance at 25 and 50% in vaseline was applied to the clipped skin of 5 animals for 48 h using an occlusive dressing within the preliminary experiment. Since the 50% concentration of the test substance was sufficient to cause skin irritation, this concentration was selected for topical application in the induction phase of the main experiment. For challenge exposure, intradermal injections with Freund’s adjuvant followed by epicutaneous application of 0.2 g of the test substance at concentrations of 25 and 50% in vaseline were performed in the preliminary test. After 24-h exposure under occlusive conditions, slight erythema was observed on the skin of 2/5 animals. Thus, the test substance at 25% in Vaseline was chosen for topical application in the challenge phase of the main test. In the induction phase of the main study, intradermal injections of the test substance at 1% in paraffin and/or FCA were applied into the clipped skin area of 20 females. A control group, consisting of 20 females, was injected with vehicle only and/or FCA. On Day 8, a 48-hour epicutaneous induction treatment with test substance at 50% in vaseline or vehicle only was performed in the treated or control animals on the regions of intradermal injections. On Day 22, the challenge treatment was performed by topical application of the test substance at 25% concentration in vaseline and the vehicle to all animals for 24 h. Skin reactions were evaluated 24 and 48 h after the challenge application. During the study no test substance-related clinical signs and no effects on body weight gain were observed. No cutaneous reactions were provoked by the challenge treatment with the test substance at 25% in none of the animals of the test and control groups. Therefore, the test substance had no sensitising effect in guinea pigs under the experimental conditions chosen.

Human data

Triglycerides, mixed decanoyl and octanoyl (CAS 73398 -61 -5) was tested for its skin sensitisation potential in a human Repeated Insult Patch Test (Eisenberg, 1996). 54 volunteers were induced epicutaneously (interscapular back skin) with 0.2 mL of the unchanged test substance for an exposure period of 24 h under semi-occlusive conditions. Inductions were performed three times a week for a total of ten applications. The challenge application was performed two weeks after the last induction on the volar forearm. Skin reactions were observed 24 and 48 h after challenge application on the volar forearm. None of the 54 tested volunteers showed any skin reactions, indicating that the test material was not skin sensitising to human skin under the conditions of this study.

Overall conclusion for skin sensitisation

A weight-of-evidence approach was applied to assess the skin sensitising potential of the registration substance Glycerol monomyristate (CAS 27214 -38 -6). The skin sensitisation studies and/or human data with three source substances were all negative. Taking into account the available information, Glycerol monomyristate (CAS 27214 -38 -6) is not expected to be skin sensitising.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that "substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Glycerol monomyristate, data will be generated from data available for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

The available data on skin sensitisation do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.