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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:
31 October - 23 December 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD guideline 406 without any deviation

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
dated 17 July 1992
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
dated 31 July 1992
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
At the time of study completion (1996), the LLNA method was not adopted.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
whitish powder
Details on test material:
- Storage condition of test material: Room temperature
- Manufacturing date: February 1995
- Expiration date: February 1997
- Purity test date: 30 October 1996
Specific details on test material used for the study:
Date of receipt: 05 November 1996
Name as cited in the report: AMITROLE

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Centre d'Elevage Lebeau, 78950 Gambais, France
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: ca. 3 months
- Weight at study initiation: Test group: 340 ± 22 g for males, 416 ± 17 g for females; control group: 310 ± 13 g for males, 340 ± 37 g for females
- Housing: Animals were housed in inox cages in groups of 5 animals of the same sex.
- Diet: 106 diet (U.A.R., 91360 Villemoisson-sur-Orge, France), ad libitum
- Water: Drinking water filtered by a F.G. Millipore membrane (0.22 micron), ad libitum
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 30-70%
- Air changes: 12 per hour
- Photoperiod: 12 hours light / 12 hours dark

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
10% w/v in vehicle or in a 1:1 mixture (v/v) FCA/physiological saline
Day(s)/duration:
on Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
on Day 8
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
on Day 22
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
on Day 36
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Range finding test: 2 females in intradermal exposure, and 2 females in topical exposure
Main test: 10 (5/sex) and 20 (10/sex) animals in control and test groups, respectively
Details on study design:
RANGE FINDING TESTS:
- Intradermal route: Considering the maximal practicable concentration of 25% (w/w), two female Guinea pigs received intradermal injections (0.1 mL) of the test item at concentrations of 5, 10 or 25% w/v in vehicle; and observed for skin irritation reactions at 24 and 48 hours and 6 days after injection.
- Topical route: Two female guinea pigs were applied with undiluted test material under occlusive dressings for 24 hours and observed for skin reactions at 24 and 48 hours after removal of the dressings.


MAIN STUDY
A. INDUCTION EXPOSURE: INTRADERMAL
- On Day 1, six injections (0.1 mL) were made into the dermis of a clipped area in the dorsal region between the shoulders (3 injections on each side).
- Test group: Intradermally injected with FCA diluted at 50% (v/v) with saline (0.9% NaCl); test item at 10% (w/w) in vehicle (sterile isotonic saline); and 10% test item in a 1:1 mixture (v/v) of FCA/saline.
- Control group: Intradermally injected with FCA diluted at 50% (v/v) with saline; saline only; and 1:1 mixture (v/v) of FCA/saline.

B. INDUCTION EXPOSURE: TOPICAL
- On Day 7, the scapular area was clipped. As the test substance was shown to be non-irritant during the preliminary tests, the animals were treated with 0.5 mL of sodium lauryl sulfate (10% w/w) in Vaseline in order to induce local irritation.
- On Day 8, undiluted test material (0.5 g) was topically applied for 48 hours via occlusive patch to the region of the intradermal injections in test groups. In control group, only vehicle (0.5 mL) was topically applied on Day 8 via occlusive patch.
- Cutaneous reactions were recorded one hour after removal of the occlusive dressing on Day 10.

C. CHALLENGE EXPOSURE: TOPICAL
- On Days 22 and 36 (1st and 2nd challenges), undiluted test material (0.5 g) was topically applied for 24 hours via occlusive patch to the posterior right flank and 0.5 mL of the saline was applied to the posterior left flank in both test and control groups.
- After 1st challenge, cutaneous reactions were recorded on Days 24, 25 and 26 and animals were sacrificed on Day 35.
- After 2nd challenge, cutaneous reactions were recorded on Days 38 and 39. Animals were sacrificed on Day 39 and skin samples were isolated.


OTHER:
- Clinical examinations: Animals were observed twice a day during the study in order to check for clinical signs and mortality.
- Body weight: Animals were weighed individually on the day of allocation into the groups, on Days 1, 8, 15 and 25 (or 35) and on the last day of the study.
- Pathology: No necropsy or histological examination was performed. At the end of the study, isolated skin samples were preserved in 10% buffered formalin.
Challenge controls:
A group of 10 animals (5/sex) was intradermally induced with FCA and/or vehicle on Day 1; topically induced with vehicle on Day 8 and topically challenged with undiluted test item on Day 22.
For second challenge application on Day 36, ten untreated animals (naïve animals) were topically challenged with undiluted test item.
Positive control substance(s):
yes
Remarks:
historical data: 2,4-dinitrochlorobenzene

Results and discussion

Positive control results:
Historical data (June 1996): 2,4-dinitrochlorobenzene (0.1% intradermal induction, 1% topical induction and 0.5% topical challenge) induced positive reactions in 50% Guinea pigs

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
72
Group:
test group
Dose level:
100%
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
no deaths or clinical signs of toxicity were noted
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
3
Total no. in group:
20
Clinical observations:
no deaths or clinical signs of toxicity were noted
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

PRELIMINARY STUDY

- Following intradermal injections, at 24 and 48 hours and 6 days, irritation was noted at 5 and 10% test item concentrations and necrosis was noted at 25% test concentration. Therefore, 10% concentration was selected for intradermal induction for the main study.

- Following topical applications, no skin irritation reaction was noted in any animal at 24 and 48 hours after removal of the dressings. Therefore 100% concentration was selected for topical induction and challenge applications for the main study.

MAIN STUDY

- No clinical signs and no deaths were noted during the study.

- Body weight gain of the treated animals was normal when compared to that of the control animals.

- After the first challenge application, no cutaneous reactions were observed in the control animals. Only dryness of the skin was noted in one animal at the 48 and 72-hour readings.

- In the treated group, at the 24-hour reading, a very slight or well-defined erythema was noted in 7/20 and 3/20 animals, respectively. At the 48-hour reading, a very slight or well-defined erythema persisted in 7/20 and 3/20 animals, respectively. Dryness of the skin was noted in 6/20 animals, and crusts were noted in one animal. At the 72-hour reading, a very slight or well-defined erythema persisted in 2/20 and 1/20 animals, respectively. Dryness of the skin was noted in 8/20 animals, and crusts were noted in one animal. No oedema was observed.

- After the second challenge application, very slight or well-defined erythema was observed in 4/20 and 3/20 animals of the treated group, 24 hours after removal of the pads. At the 48-hour reading, a very slight erythema persisted in 5/20 animals, and dryness of the skin was noted in 6/20 animals. No oedema was observed.

- No cutaneous reactions were noted in the naive control animals.

- After the first cutaneous challenge application, the observed cutaneous reactions were attributed to a sensitizing effect of the test substance in 4/20 animals (score of erythema >=2). This result was confirmed after the second challenge application (positive reaction in 3/20 animals).

- Refer tables 7.4.1/1 and 7.4.1/2 for individual animal scores

Table 7.4.1/1: Skin reactions after first challenge

 

Sex

Animal no.

Day 24 (24 hours)

Day 25 (48 hours)

Day 26 (72 hours)

Erythema

Oedema

Erythema

Oedema

Erythema

Oedema

LF

RF

LF

RF

LF

RF

LF

RF

LF

RF

LF

RF

Male

1

0

0

0

0

0

0

0

0

0

0

0

0

2

0

1

0

0

0

1/S

0

0

0

0/S

0

0

3

0

0

0

0

0

0

0

0

0

0

0

0

4

0

0

0

0

0

0

0

0

0

0

0

0

5

0

1

0

0

0

1/S

0

0

0

0/S

0

0

6

0

2

0

0

0

1/S

0

0

0

1/S

0

0

7

0

1

0

0

0

1

0

0

0

0

0

0

8

0

2

0

0

0

2/S/A

0

0

0

2/S/A

0

0

9

0

1

0

0

0

1

0

0

0

0/S

0

0

10

0

0

0

0

0

0

0

0

0

0

0

0

Female

1

0

0

0

0

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

0

0

0

0

3

0

1

0

0

0

0

0

0

0

0

0

0

4

0

0

0

0

0

0

0

0

0

0

0

0

5

0

0

0

0

0

0

0

0

0

0

0

0

6

0

1

0

0

0

1

0

0

0

0

0

0

7

0

0

0

0

0

1

0

0

0

0/S

0

0

8

0

0

0

0

0

0

0

0

0

0

0

0

9

0

1

0

0

0

2/S

0

0

0

1/S

0

0

10

0

2

0

0

0

2/S

0

0

0

0/S

0

0

LF: Left flank (control); RF: right flank (treated); S: dryness of the skin; A: crusts

 

Table 7.4.1/2: Skin reactions after second challenge

 

Sex

Animal no.

Day 24 (24 hours)

Day 25 (48 hours)

Day 26 (72 hours)

Erythema

Oedema

Erythema

Oedema

Erythema

Oedema

LF

RF

LF

RF

LF

RF

LF

RF

LF

RF

LF

RF

Male

1

0

0

0

0

0

0

0

0

0

0

0

0

2

0

0

0

0

1

0

0

0

1

0

0

0

3

0

0

0

0

0

0

0

0

0

0

0

0

4

0

0

0

0

0

0

0

0

0

0

0

0

5

0

0

0

0

0

0

0

0

0

0

0

0

6

0

0

0

0

1

0

0

0

0/S

0

0

0

7

0

0

0

0

0

0

0

0

1/S

0

0

0

8

0

0

0

0

2

0

0

0

1/S

0

0

0

9

0

0

0

0

0

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

0

0

0

0

0

Female

1

0

0

0

0

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

0

0

0

0

0

4

0

0

0

0

0

0

0

0

0

0

0

0

5

0

0

0

0

0

0

0

0

0

0

0

0

6

0

0

0

0

1

0

0

0

0

0

0

0

7

0

0

0

0

2

0

0

0

1/S

0

0

0

8

0

0

0

0

0

0

0

0

0

0

0

0

9

0

0

0

0

2

0

0

0

1/S

0

0

0

10

0

0

0

0

1

0

0

0

0/S

0

0

0

LF: Left flank (control); RF: right flank (treated); S: dryness of the skin

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under these test conditions, the test item is not classified for skin sensitization according to CLP Regulation (EC) N° (1272-2008) and according to the Globally Harmonised System of classification and labelling of chemicals (GHS). No hazard statement or signal word is required.
Executive summary:

In a Magnusson & Kligman maximisation study (GPMT) performed according to OECD guideline 406 and in compliance with GLP, 20 Dunkin Hartley guinea pigs (10/sex) were intradermally induced with three injections of 0.1 mL of FCA, 10% test item in saline and 10% test item emulsion in FCA on Day 1 on shoulder region on each side of mid-line. On Day 7, the animals were treated with 0.5 mL of sodium lauryl sulfate (10% w/w) in vaseline in order to induce local irritation. On Day 8, the same area on which intradermal injections were made was topically induced with 0.5 g of undiluted test item via occluded patch for 48 hours. On Day 22 and 36, 24-hour occlusive challenge patches of 0.5 g undiluted test item or 0.5 mL of saline was applied to right or left flank, respectively. A control group of 10 animals (5/sex) was intradermally injected with FCA, saline or FCA emulsion in saline on Day 1; topically induced with saline on Day 8 and topically challenged with undiluted test item on Day 22. For second challenge application on Day 36, ten untreated animals (naïve animals) were topically challenged with undiluted test item. The test concentrations for the main study were determined from a preliminary study using two animals.

No clinical signs and no deaths were noted during the study. After the first challenge application, no cutaneous reactions were observed in the control animals. Only dryness of the skin was noted in one animal at the 48 and 72-hour readings. In the treated group, at the 24-hour reading, a very slight or well-defined erythema was noted in 7/20 and 3/20 animals, respectively. At the 48-hour reading, a very slight or well-defined erythema persisted in 7/20 and 3/20 animals, respectively. Dryness of the skin was noted in 6/20 animals, and crusts were noted in one animal. At the 72-hour reading, a very slight or well-defined erythema persisted in 2/20 and 1/20 animals, respectively. Dryness of the skin was noted in 8/20 animals, and crusts were noted in one animal. No oedema was observed. After the second challenge application, very slight or well-defined erythema was observed in 4/20 and 3/20 animals of the treated group, 24 hours after removal of the pads. At the 48-hour reading, a very slight erythema persisted in 5/20 animals, and dryness of the skin was noted in 6/20 animals. No oedema was observed.

No cutaneous reactions were noted in the naive control animals. After the first cutaneous challenge application, the observed cutaneous reactions were attributed to a sensitizing effect of the test substance in 4/20 animals (score of erythema >=2). This result was confirmed after the second challenge application (positive reaction in 3/20 animals).

The sensitivity of the guinea-pigs was satisfactory since 50% of the animals showed a positive reaction with positive control 2,4-dinitrochlorobenzene.

Under these test conditions, the test item is not classified for skin sensitization according to CLP Regulation (EC) N° (1272-2008) and according to the Globally Harmonised System of classification and labelling of chemicals (GHS). No hazard statement or signal word is required.