Myristica fragrans, ext.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 October 1984 - 12 November 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted according to methods resembling those outlined in OECD guideline 406 and under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

4 animals in the control group, slightly different scoring system used

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

This reliable study was already conducted in 1992 and the outcome is considered relevant for this endpoint.

Test material

Test material information

Constituent 1

Reference substance name:

84-209-1

IUPAC Name:

84-209-1

Constituent 2

Reference substance name:

Nutmeg Oil EI

IUPAC Name:

Nutmeg Oil EI

Details on test material:

- Name of test material (as cited in study report): 84-209-01, Nutmeg Oil EI
- Physical state: Liquid

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Hazelton Research Animals, Inc., Denver, Pennsylvania, USA
- Age at study initiation: No data
- Weight at study initiation: 300-500 g
- Housing: Two per cage, in accordance with guide(line)
- Diet (e.g. ad libitum): Ad libitum, guinea pig diet
- Water (e.g. ad libitum): Ad libitum, fresh tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: 0.9% saline (intradermal) and 80% ETOH (epicutaneous)

Induction 2

Concentration / amount:

Intradermal induction: 1.0%
Epicutaneous induction: 4.0%
Epicutaneous challenge: 2.0%

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: 0.9% saline (intradermal) and 80% ETOH (epicutaneous)

Challenge 2

Concentration / amount:

Intradermal induction: 1.0%
Epicutaneous induction: 4.0%
Epicutaneous challenge: 2.0%

No. of animals per dose:

20 (10 males, 10 females)

Details on study design:

RANGE FINDING TESTS:
Intradermal irritation potential of test articles was determined in 4 guinea pigs. These were exposed to 6 different concentrations intradermal (0.1%, 0.5%, 1.0%, 1.5%, 3.0% and 5.0%) and reactions were scored after 24 hours. Topical irritation potential was determined in 10 guinea pigs exposed to 12 different concentration (0.5%, 1.0%, 1.5%, 2.0%, 4.0%, 6.0%, 8.0%, 15%, 25%, 35% and 45% and reactions were scored 24 hours after removal.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous)
- Exposure period: 48 hours (epicutaneous)
- Test group (n=20): Test article, applied according to guideline (7 days between intradermal and epicutaneous induction)
- Negative control group (n=4): Only vehicle, applied according to guideline (7 days between intradermal and epicutaneous induction)
- Positive control group (n=6): 0.1% DNCB, applied according to guideline (7 days between intradermal and epicutaneous induction)
- Site: Shoulders, nearest to the head
- Frequency of applications: Once (intradermal and epicutaneous)

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Approx. 14 days after epicutaneous induction
- Exposure period: 24 hours
- Test groups: Both test article and vehicle were applied
- Negative control group: Both test article and vehicle were applied
- Positive control group: Both 0.1% DNCB and vehicle were applied
- Site: Left (test article) and right (vehicle) flank
- Evaluation (hr after challenge): 24 and 48 hours

C. SCORING SYSTEM
0 - No reaction
1 - Slight patchy mild redness
2 - Moderate and diffuse redness
3 - Intense redness and swelling
4 - Severe erythema and edema; skin damage

Scoring system was used to calculate the sensitization rate. Additionally, Kligman’s classification scheme modified to reflect a treatment group of twenty animals was used for ranking the substances in order of their sensitization capacity (see table below). According to the percentage of animals sensitized, the substance was assigned to one or another of five classes, ranging from weak (grade I) to extreme (grade V), regardless of the intensity of the response.

Modified Maximisation Grading
Sensitization
Percent Animals Grade Classification
0-8 1-2 I Weak*
9-28 3-6 II Mild
29-64 7-13 III Moderate
65-80 14-16 IV Strong
81-100 17-20 V Extreme
*Magnusson and Kligman (1969) do not regard Sensitization of Grade ! as significant

Challenge controls:

Not performed

Positive control substance(s):

yes

Remarks:

0.1% 1-chloro-2,4-dinitrobenzene (DNCB, n=6)

Results and discussion

Positive control results:

A positive response was elicited in the positive control group (n=6) when challenged with 0.1% DNCB. At both readings a sensitisation rate of 83% was calculated.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

4.0% and 2.0% test article

No. with + reactions:

9

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 4.0% and 2.0% test article. No with. + reactions: 9.0. Total no. in groups: 20.0.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

4.0% and 2.0% test article

No. with + reactions:

11

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 4.0% and 2.0% test article. No with. + reactions: 11.0. Total no. in groups: 20.0.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

vehicle and 2.0% test article

No. with + reactions:

0

Total no. in group:

4

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: vehicle and 2.0% test article. No with. + reactions: 0.0. Total no. in groups: 4.0.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

vehicle and 2.0% test article

No. with + reactions:

0

Total no. in group:

4

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: vehicle and 2.0% test article. No with. + reactions: 0.0. Total no. in groups: 4.0.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.1% DNCB

No. with + reactions:

5

Total no. in group:

6

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.1% DNCB. No with. + reactions: 5.0. Total no. in groups: 6.0.

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.1% DNCB

No. with + reactions:

5

Total no. in group:

6

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.1% DNCB. No with. + reactions: 5.0. Total no. in groups: 6.0.

Any other information on results incl. tables

No mortality and effect on body weight were reported.

Calculated sensitisation rates:

- Test group, 1st reading: 45%

- Test group, 2nd reading: 55%

- Negative control group, 1st reading: 0%

- Negative control group, 2nd reading: 0%

Test article was indicated as Grade III (= moderate) sensitiser in the study report, based on Magnusson and Kligman (1969) sensitisation rate grading.

Individual animal scores are reported in the table below.

Individual Animal Scores after Challenge
		24 hours		48 hours
Animal #	Sex	Left	Right	Left	Right
5351	M	0	0	0	0
5352	M	2	0	2	0
5353	M	2	0	2	0
5354	M	0	0	1	0
5355	M	2	0	2	0
5356	M	2	0	2	0
5357	M	2	0	2	0
5358	M	3	0	3	0
5359	M	2	0	2	0
5360	M	0	0	0	0
5361	F	1	0	2	0
5362	F	0	0	0	0
5363	F	0	0	0	0
5364	F	0	0	0	0
5365	F	0	0	0	0
5366	F	2	0	2	0
5357	F	0	0	0	0
5358	F	0	0	1	0
5359	F	0	0	0	0
5370	F	0	0	0	0
Total number of animals responding		9	0	11	0

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information

Conclusions:

Under the conditions of this study, a postive skin response was observed in guinea pigs exposed to a challenge concentration of 2.0%. The sensitisation rate was calculated to be at least 45%, which exceeds the threshold value of 30% for classification. Therefore, the substance needs to be classified as sensitising based on the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.

Executive summary:

This guinea pig maximisation test (GPMT) was performed to determine the sensitising potential of Nutmeg Oil EI. In the test group (n=20, M:10, F:10), intradermal induction was performed with 1.0% test article, epicutaneous induction with 4.0% test article and the final challenge exposure was done with 2.0% test article. Negative (n=4) and positive (n=6) control groups were also included. Mortality and body weights were recorded.

No mortality and effect on body weight were reported. In the test group, 9 and 11 out of 20 guinea pigs (at 1st and 2nd reading, respectively) showed a positive skin reaction after challenge exposure to Nutmeg Oil EI. No positive skin reactions were noted in the negative control group. The results in the positive control group confirmed validity of the test (system), with 5 out of 6 animals showing positive skin reactions at the 1st and 2nd reading after challenge exposure (to 0.1% DNCB).

Under the conditions of this study, a postive skin response was observed in guinea pigs exposed to a challenge concentration of 2.0%. The sensitisation rate was calculated to be at least 45%, which exceeds the threshold value of 30% for classification. Therefore, the substance needs to be classified as sensitising based on the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.