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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 28, 2013 to December 28, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD test guidelines in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
Data are available to the registrant from existing data prepared using the OECD 406 Beuhler sensitisation test method. The data are considered suitable to assess the senstisation potential of the substance and repeat animal testing is considered not justifiable.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Clear colorless liqu id
Details on test material:
Name: DEGMEE
CAS No.: 1002-67-1
Lot No.: 20130625
Storage condition: Room temperature [(1 ~ 30) °C]
Expiration date: 2014-06-25
Appearance: Clear colorless liquid
Purity: 99.98 %
Molecular formula / MW: C2H5O(CH2CH2O)CH3 / 148.20

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
Animal strain, species and sex: ElmSam:HA, Guinea pig, Male, SPFSupplier: Samtako Bio Korea Co., Ltd. (77- 1, Seorang-dong, Osan-si, Gyeonggi- do, Korea)Number of animals: receipt - 50 animals, treatment - 8 animals (preliminary study), 40 animals (Main study)Age at start of treatment: approximately 6 weeks oldBody weight at the time of allocation: 329.94 ~ 378.36 g (Main study)Choice of test system: The Hartley guinea pig is a preferred species of choice as used for predictive sensitisation studies for several decades and accumulated enough raw data to be compared and easy to be interpreted of experimental results.Quarantine and acclimation: The animals were examined for quarantine on the receipt and acclimatized to the laboratory conditions for 7 days. Healthy animals were selected and used for the study based on general health conditions.Identification: Picric acid was used for individual recognition. And a cage card was attached to each cage for recognition as follows. The cage card had information including the study number, test substance name, test item, animal receipt date, acclimation period, animal allocation date, study period, sex/animal number and name of study director.Animal allocation: After quarantine and acclimation period, the animals were weighed and located using programme of SOP of Korea Testing & Research Institute.Remained animals: After animal allocation, remained animals were euthanized.Environmental conditions: The animal facility was maintained at (20 ± 3) °C, relative humidity of (50 ± 20)%, air ventilation of (10 ~ 15) changes/hr. 12 hours light/dark cycle (light during 8:00 ~ 20:00) at (150 ~ 300) Lux.Environmental monitoring: The temperature and relative humidity were monitored automatically every half-hour by automatic instrument and other environmental condition (illuminance, ammonia concentration, noise of animal room, etc) were measured periodically (1 time/quarter) by SOP of Korea Testing & Research Institute. There were no influenceable variations for study in environmental measurements.Housing: Five animals per cage were housed in stainless steel cages [700(W) mm x 475(0) mm x 200(H) mm, Daejong Instrumental Industry Co., Ltd, Korea] for the study periods including quarantine and acclimation.Diet and water: The animals were fed pellet diet for experimental guinea pig including 0.01 % ascorbic acid (Cargil Agri Purina, Inc. 56-4, Soryong-dong, Gunsan-si, Jeollabuk-do, Korea) and given the filtered and UV irradiated water ad libitum.Contaminant confirmation of diet and water: The diet was considered not to contain any contaminant based on the periodical analysis results report of manufacturer and water did not contain any contaminant based on periodical analysis in accordance with the SOP of Korea Testing &Research Institute.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% test substanceFurther details can be found in Any other information
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100% test substanceFurther details can be found in Any other information
No. of animals per dose:
20 - treatment & 10 for each control groupFurther details can be found in Any other information
Details on study design:
Preliminary testDose levels: The concentrations of test substance were 4 levels, 50%(v/v), 25%(v/v), 12.5%(v/v) with vehicle and including the highest concentration [100%; test substance itself] by using a common ratio 2.Test: The one flank for 2 animals per concentration was closely clipped [(4 x 6)cm2] with hair clipper before the treatment day. The patch [(2.5 x 2.5)cm2, Aceband- S, Youngchemical Co. Ltd., Korea] was fully-loaded with test substance (0.5 ml) and applied to the clipped region and maintained using elastic bandage (Coban, 3M, USA) for 6 hours.Determination of the dose levels: In the preliminary test using 2 animals per concentration, skin reaction were not observed at 100% concentration (test substance itself). Based on this result, concentration of 100% concentration was chosen for the induction and challenge phase of the main test.Main testInduction exposure: Day 0, 7, 14The fur on one flank [(4 x 6)cm2] was closely clipped with a hair clipper before the treatment day. On the treatment day, 0.5 ml each of the test substance, absorbed in a patch [(2.5 x 2.5)cm2, Aceband-S, Youngchemical Co., Ltd., Korea] was attached to cover the test area and maintained using elastic bandage (Coban, 3M, USA) for 6 hours. In the control groups, D.W. or 1%(w/v) DNCB was applied in a similar manner to the test area. The same application as on day 0 was carried out on the same test area of the same flank on day 7 and again on day 14.Challenge exposure: Day 28The untreated flank of treated and control animals were clipped on the day prior to the challenge patch application. To the treated and negative control animals, patches [(2.5 x 2.5)cm2, Aceband- S, Youngchemical Co., Ltd., Korea] which absorbed 0.5 ml of the test substance were attached to the test area and maintained using elastic bandage (Coban, 3M, USA) for 6 hours. In the positive control group, 1%(w/v) DNCB was applied in a similar manner to the test area.ObservationsClinical signs: All the animals were daily observed for clinical signs and survival during the experimental period.Measurement of body weight: Individual body weight was measured on the animal receipt day, animal allocation day and once a week until the end of test.Observation of skin: Approximately 21 hours after removing the patch of the challenge area, the sites were wiped gently with gauze, and the challenged sites and surrounding area were shaved. At 3 hours later, the skin reaction was observed according to the grades as described in Table (see Any other information). The second observation was made at 24 hours after the first evaluation.Evaluation of skin reactionSkin reaction: The skin reactions were observed and recorded according to the grades shown in "[Table] Magnusson and Kligman grading scale" (see Any other information).At evaluation, grades of 1 or greater in the test substance group generally indicate sensitisation, provided grades of less than 1 are seen in negative control animals. If grades of 1 or greater are noted in negative control animals, then the reactions of test animals which exceed the most severe reaction in control animals are presumed to be due to sensitisation.Evaluation: The outcome of the test was presented as sensitisation index (mean score of skin reaction) and frequency index (sensitisation rate) in test and control animals and evaluated according to the grades shown as "[Table] Evaluation standard" (see Any Other information).Sensitisation Index (the mean score of skin reaction)= Σ Score of Grading scale / Number of animalsFrequency Index (sensitisation rate, %)= [Number of animals with skin reaction / Number of animals] x 100Statistics of data: The body weight was presented as mean ± S.D. The one way ANOVA test was conducted to determine which pairs of group comparison were significantly different by using SPSS programme (ver. 19).
Challenge controls:
Details can be found in Any other information
Positive control substance(s):
yes
Remarks:
1-chloro-2,4-dinitrobenzene (DNCB)

Results and discussion

Positive control results:
In the positive control group, skin reaction of erythema was shown and sensitisation index (the mean score of skin reaction) was '1.6 (24 hours)' and '0.6 (48 hours)'. Also, the frequency index (sensitisation rate) was '100.0% (24 hours)' and '60.0% (48 hours)'.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
100% test substance
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100% test substance. No with. + reactions: 0.0. Total no. in groups: 20.0.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
100% test substance (challenge exposure)
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100% test substance (challenge exposure). No with. + reactions: 0.0. Total no. in groups: 10.0.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
1% (w/v) DNCB
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 1% (w/v) DNCB. No with. + reactions: 10.0. Total no. in groups: 10.0.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
1% (w/v) DNCB
No. with + reactions:
6
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 1% (w/v) DNCB. No with. + reactions: 6.0. Total no. in groups: 10.0.

Any other information on results incl. tables

Mortality and clinical signs

Group

Number of animals

Mortality (dead/total)

Clinical signs

G1

20

1/20a

Moribunda

NAD

G2

 

10

0/10

NAD

G3

 

10

0/10

NAD

NAD: No Abnormalities Detected

a: One animal was moribund with right hind limb caught in bottom of the cage on 1 day after 1stinduction treatment and was euthanized (Animal No.: 1108)

 

Body weight                                      Unit: g

Group

 

Day(s) after treatmenta

-1b

6

13

20

27

G1

Mean

S.D.

N

353.41

9.21

20

408.20

14.83

19c

457.57

22.55

19c

510.62

25.41

19c

581.85

33.59

19c

G2

Mean

S.D.

N

352.52

12.65

10

399.30

25.92

10

445.36

42.05

10

495.60

46.77

10

550.28

53.57

10

G3

Mean

S.D.

N

352.88

9.83

10

417.36

16.29

10

476.89

23.62

10

538.63

40.21

10

610.05

42.26

10

a: 1stinduction,b: Body weight at the time if allocation, S.D.: standard deviation

N: Number of Animals

c: One animal was moribund with right hind limb caught in bottom of the cage on 1 day after 1stinduction treatment and was euthanized (Animal No.: 1108)

 

Evaluation of skin reaction

Group

Number of animals

Observation time

Score of skin reaction

Mean score

Sensitisation rate (%)

0

1

2

3

G1

19a

24

48

19*

19

0

0

0

0

0

0

0.0

0.0

0.0

0.0

G2

10

24

48

10

10

0

0

0

0

0

0

0.0

0.0

0.0

0.0

G3

10

24

48

0

4

4

6

6

0

0

0

1.6

0.6

100.0

60.0

*: Number of animals showing the score

a: One animal was moribund with right hind limb caught in bottom of the cage on 1 day after 1stinduction treatment and was euthanized (Animal No.: 1108)

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The results indicated that this test substance is considered to have no skin sensitizing potency of DEGMEE in guinea pigs by Buehler test under the conditions of this study.
Executive summary:

This study was conducted to evaluate the skin sensitisation of DEGMEE in guinea pigs by Buehler test. The study was conducted in accordance with the following test regulation:

OECD Guidelines for the testing of chemicals, Section 4, TG. No. 406 ‘Skin Sensitisation’ (July 17, 1992)

 

Parameters measured during the study period were mortality, clinical signs, body weight changes and skin reactions. The following results were obtained.

- No treatment-related mortality was observed during the study period.

- No treatment- related clinical signs were observed in any treated animals.

- All living animals showed a normal increase of body weight.

- At the 24 and 48 hours after challenge with test substance of 100% concentration, the sensitisation index (mean score of skin reaction) and frequency index (sensitisation rate) were '0.0', '0.0' and '0.0%', '0.0%' at 24 and 48 hours for the test substance. In positive control, the sensitisation index (mean score of skin reaction) and frequency index (sensitisation rate) were '1 .6', '0.6' and '1 00.0%', '60 .0%' at 24 and 48 hours after challenge, respectively.

 

These results indicated that the test substance, DEGMEE, was considered to have no skin sensitizing potency in guinea pigs by Buehler test under the conditions of this study.