1-O-β-(2 ,3-di-O-alka(e)noyl-6 -O-acetyl-D-mannopyranosyl)-D-erythritol

Endpoint summary

Description of key information

Skin Sensitisation: Not sensitising to the skin; OECD 406- Buehler Test. F. Shibata MS. (2019).

Skin Sensitisation: Not sensitising to the skin of human volunteers; RIPT 2019. V. Pessoto Rosa (See Section 7.10.4)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

9th January 2008 - 28th February 2008

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Only half of the animals numbered required by OECD 406 were used and the study was not conducted to GLP standard. No positive control groups was included. Temperature and humidity were outside of guideline range.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

1992

Deviations:

yes

Remarks:

Buehler guideline requires 20 animals/groups but only 10 tested and without positive control groups. Temperature and humidity were also outside of guideline range.

GLP compliance:

no

Remarks:

The experiment was performed in between January and March 2008, therefore pre-dating 1st June 2008 cut off after which GLP compliance is necessary for toxicological tests. The report is dated 2019, because this is when the report itself was finalised.

Type of study:

Buehler test

Justification for non-LLNA method:

This is an existing study for the sensitisation endpoint (study performed January - March 2008).

Species:

guinea pig

Strain:

Hartley

Remarks:

Slc:Hartley [SPF]

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Kitayama Labes Co., Ltd..
- Females (if applicable) nulliparous and non-pregnant: Not stated
- Age at study initiation: 6 weeks at preliminary study and 7 weeks at induction
- Weight at study initiation: 350 – 411 g
- Fasting period before study: yes (17 hour)
- Housing: Animals were individually housed in wire mesh cage, 30.5W×49.5D× 33.5H (cm), One animal/cage (during the study).
- Diet (e.g. ad libitum): RC4(Oriental Yeast, Lot No. 070607 and 0171119)
- Water (e.g. ad libitum): tap water from automated water supply system ad libitum. 
- Acclimation: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0−27.0°C
- Humidity (%): 35.0−75.0%
- Air changes (per hr): 8 or more times air changes per hour
- Photoperiod (hrs dark / hrs light): 12 : 12
IN-LIFE DATES: 16/1/2008 – 28/2/2008


FORM AS APPLIED IN THE TEST (if different from that of starting material): Pale yellow liquid

OTHER SPECIFICS: N/A

Route:

epicutaneous, open

Vehicle:

other: Ethanol

Remarks:

1.5mL test item diluted with ethanol to 3mL (a 50 v/v% solution)

Concentration / amount:

0.2 mL formulation

Day(s)/duration:

6-hour

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, open

Vehicle:

other: Ethanol

Remarks:

1.0 mL test item diluted to 5.0 mL (a 20 v/v% solution)

Concentration / amount:

0.2 mL

Day(s)/duration:

6-hours

Adequacy of challenge:

not specified

No. of animals per dose:

Preliminary irritation testing: 2
Test group: 10
Sham group: Not included
Naive control group: 5

Details on study design:

RANGE FINDING TESTS:
Two animals were treated with 3 occlusive patches containing 50, 20 or 10% test item in ethanol for 6 hours. The application sites were observed at 24hours after removal of patches. As the results, only 50% v/v induced discrete or patchy erythema at the treatment sites, and no animal showed unusual systemic reactions during the observation period. Therefore, 50 and 20% v/v were selected for the induction and challenge exposure respectively.

MAIN STUDY
A. INDUCTION EXPOSURE
The hair on the skin of induction site of each animal was removed with an electric clipper and shaver. A 0.2 mL aliquot of the formulation or ethanol was applied to each induction site. After 6-hour application, these substances were removed. This application was conducted 3 times in total every seven days.

B. CHALLENGE EXPOSURE
On the 14th day after the last induction, hair on the challenge site of each animal was removed with an electric clipper and shaver. For the both test substance treatment group and negative control group, 0.2 mL each of the 20% v/v test substance formulation was applied to the challenge site. After 6-hour application the formulation was removed.

The application sites of representative animals were photographed with a digital camera at 24 hours after removal of the challenge patches. The skin reactions were observed at 24 and 48 hours after removal of the challenge patches and scored according to the skin reaction grading scale below;

No visible change = 0
Discrete or patch erythema = 1
Moderate and confluent erythema = 2
Intense erythema and swelling = 3

After scoring the skin reactions, the highest score of each animal was selected as the evaluation score for the animals. When an animal of the test substance treatment group showed the higher score than the highest individual score in the negative control group, this test substance treatment anima was judge to be positive for sensitisation. The skin reaction ratio was calculated according the following equation;
Skin sensitisation ratio = [(number of animals positive for sensitisation /number of animals tested) x 100].

The general condition including the skin reactions at the induction sites was observed once daily except for holidays until the euthanasia.

Challenge controls:

Negative control group receiving ethanol only.

Positive control substance(s):

no

Positive control results:

No positive control group was included in the study

Key result

Reading:

other: Skin sensitisation ratio (%)

Hours after challenge:

48

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No abnormality observed

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

other: Skin sensitisation ratio (%)

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No abnormal observation

Remarks on result:

no indication of skin sensitisation

Table 3. Skin sensitisation ratio in Buehler test

Group	Concentration of compound (%)*		Number of animals	Challenge
Test substance	Induction	Challenge		Minimum score	Maximum score	Skin sensitisation ratio (%)
	50	20	10	0	0	0
Negative Control	0	20	5	0	0	-

* Vehicle: Ethanol (76.9 -81.4 vol%)

Grading scale: 0 -3

-: Not applicable

Table 4. Individual daily score for skin sensitisation

Group	Concentration of compound (%)*		Animals ID No.	Time after patch removal of challenge (hr)
	Induction	Induction		24	48
Test substance	50	20	1	0	0
			2	0	0
			3	0	0
			4	0	0
			5	0	0
			6	0	0
			7	0	0
			8	0	0
			9	0	0
			10	0	0
			Average	0	0
Negative control	0	20	11	0	0
			12	0	0
			13	0	0
			14	0	0
			15	0	0
			Average	0	0

*Vehicle: Ethanol (76.9 -81.4 vol%)

Grading scale: 0 -3

Table 5. Clinical observation

Group	Concentration of compound (%)*		Number of animals	Sign	Experimental week
Test substance	Induction	Challenge			1	2	3	4	5
	50	20	10	No abnormality observed	10	10	10	10	10
Negative Control	0	20	5	No abnormality observed	5	5	5	5	5

* Vehicle: Ethanol (76.9 -81.4 vol %)

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the condition of the study, no conclusion can be made on the sensitisation potential of the substance, this is due to the deviation from the OECD guideline by using only half the required animals per group and without positive control group, the study is considered inadequate for classification on the skin sensitisation for the substance, in accordance with Regulation (EC) No 1272/2008 for skin sensitisation.

Executive summary:

OECD 406 - Buehler Test (2019): A skin sensitisation study was conducted by the Buehler Test to obtain the information on delayed skin allergenic potential of the test item, using 15 female Slc:Hartley (SPF) guinea pigs.

On the basis of the results of a preliminary skin irritation study using 2 guinea pigs, 10 animals were exposed to 50% v/v of test substance-ethanol (76.9 – 81.4% vol) mixture by an occlusive patch for 6 hours 3 times at 1-week intervals, as the induction exposure. At the second week after the last induction, the test animals were exposed to 20% v/v test substance-ethanol mixture by an occlusive patch for 6 hours, as the challenge exposure.

The skin reactions were observed at 24 and 48 hours after removal of the challenge patches. For the negative control groups, 5 animals were exposed to only ethanol for induction and 20% v/v test substance-ethanol mixtures were applied to these animals for challenge.

As the results, no animal showed skin reactions in either the treatment group or the negative control group. Therefore, skin sensitisation ratio of the test substance was calculated as 0%.

From the above-described results, the test item was not considered to be a skin sensitiser in guinea pigs under the condition of this study.

However, based on the deficiencies in the study, i.e. deviation from the OECD guideline by using only half the required animals per group and without positive control group, the study is considered inadequate for classification on the skin sensitisation for the substance, in accordance with Regulation (EC) No 1272/2008 for skin sensitisation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

OECD 406 - Buehler Test (2019):

A skin sensitisation stud was conducted by the Buehler Test to obtain the information on delayed skin allergenic potential of the test item, using 15 female Slc:Hartley (SPF) guinea pigs.

On the basis of the results of a preliminary skin irritation study using 2 guinea pigs, 10 animals were exposed to 50% v/v of test substance-ethanol (76.9 – 81.4% vol) mixture by an occlusive patch for 6 hours 3 times at 1-week intervals, as the induction exposure. At the second week after the last induction, the test animals were exposed to 20% v/v test substance-ethanol mixture by an occlusive patch for 6 hours, as the challenge exposure.

The skin reactions were observed at 24 and 48 hours after removal of the challenge patches. For the negative control groups, 5 animals were exposed to only ethanol for induction and 20% v/v test substance-ethanol mixtures were applied to these animals for challenge.

As the results, no animal showed skin reactions in either the treatment group or the negative control group. Therefore, skin sensitisation ratio of the test substance was calculated as 0%.

From the above-described results, the test item was not considered to be a skin sensitiser in guinea pigs under the condition of this study.

However, based on the deficiencies in the study, i.e. deviation from the OECD guideline by using only half the required animals per group and without positive control group, the study is considered inadequate for classification on the skin sensitisation for the substance, in accordance with Regulation (EC) No 1272/2008 for skin sensitisation.

RIPT 2019 (Section 7.10.4):

Both the test product (in amount of 0.05 g/cm² of undiluted form) and control were applied to patch test filter paper discs and then applied to the right or left back (scapular area) of the study subjects (female and male, age range from 18 to 69 years old, phototype II to IV (Fitzpatrick)). The applications were performed on Mondays, Wednesdays and Fridays, during 3 consecutive weeks. Forty-eight hours (48h) after the application, the patch test was removed by trained technicians and, approximately 30 minutes after the patch test removal, the site was assessed in order to check the presence of possible clinical signs. After this period (induction) there was a, minimum, 10 day-period when no patch was applied to the study subjects' back (rest period). Then, the challenge period started. A single application of the patch test was performed, followed by readings after 48h and 72h. The study subjects were assessed by a dermatologist at the start and at the end of the study and supervised all along the study.

The study was conducted in conformance with the Declaration of Helsinque principles, the applicable regulatory requirements, including Resolution CNS no. 466/12, and in spirit of the Good Clinical Practices (Document of the Americas and ICH E6: Good Clinical Practice). Out 77 subjects, 56 subjects completed the study.

During the study, no subjects presented skin clinical signs related to the product. The product did not induce a skin sensitisation process in the study group. The product was considered safe under the study conditions. The claim “dermatologically tested” can be supported.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

MEL-B did not have any effects on p38 kinase in cultured human epidermal keratinocytes - HaCaT keratinocytes (Bae et al. 2018, see Section 7.9.4 ). p38 kinase is one of the proteins involved in skin sensitisation pathways. This finding provides further support of the negative results observed from Buehler and RIPT studies. Based on the condition of the available studies OECD 406, RIPT and cell culture study using the test item - it can be concluded the substance does not meet the requirement for classification for skin sensitisation in accordance with Regulation (EC) No 1272/2008.