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Description of key information

Skin: Under the conditions of the study, the test material is not considered to be a skin sensitiser.

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:
key study
Study period:
05 May 2000 to 16 June 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF, 59 NohSan No. 4200
Version / remarks:
1985
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study conducted prior to the adoption of the LLNA test method in 2002
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: 8 weeks (range-finding study) and 6 weeks (sensitisation study) and females: 10 to 12 weeks (range-finding study) and 8 weeks (sensitisation study).
- Weight at study initiation: males 422 to 462 g (range-finding study) and 351 to 397 (sensitisation study) and females 386 to 475 g (range-finding study) and 347 to 385 g (sensitisation study).
- Housing: The animals were housed individually in suspended stainless steel cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 13 to 21 °C
- Humidity: 47 to 77 %
- Air changes: 10 to 15 per hour
- Photoperiod: Light timers were set to maintain a 12-hour light/12-hour dark cycle
Route:
other: intradermal and topical
Vehicle:
polyethylene glycol
Remarks:
(PEG400)
Concentration / amount:
Intradermal injections (0.1mL): 1st injection pair: FCA emulsion; 2nd injection pair: 5 % (w/v) test material/PEG 400; 3rd injection pair: 5 % (w/v) test material/FCA emulsion
Topical induction (0.1mL): 10 % (w/v) test material/PEG-400
Day(s)/duration:
Intradermal induction took place on Day 0. On Day 7, animals received a topical induction application which was covered for 48 hours.
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
(PEG400)
Concentration / amount:
1.0 %, 0.3 mL
Day(s)/duration:
Day 21 for 24 hours.
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Remarks:
(PEG 400)
Concentration / amount:
0.5 or 1.0 %, 0.3 mL
Day(s)/duration:
Day 28 for 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Ten animals per sex per dose in test group, five animals per sex per dose in control groups.
Details on study design:
TOPICAL RANGE-FINDING STUDY
- Dosing: On the day prior to dose administration, four topical range-finding guinea pigs were weighed and the hair was removed from the right and left sides of the animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
- On the following day, four concentrations of the test material were prepared and 0.3 mL of each concentration was applied to the clipped area of each topical range-finding animal (1.0, 5, 10 and 20 % concentrations).
- Following chamber application, the trunk of the animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the chambers and the animal was returned to its cage. Approximately 24 hours· after chamber application, the elastic wrap, tape and chambers were removed. The test sites were then wiped with deionized water followed by dry gauze to remove test material residue. The animals were then returned to their cages.
- The test sites of the topical range-finding animals were graded for irritation at approximately 24 and 48 hours following chamber removal using the Dermal Grading System.

INTRADERMAL RANGE-FINDING STUDY
- On the day prior to dose administration, four intradermal range-finding guinea pigs were weighed and the hair removed from the. right and left sides of the animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
- On the following day, four concentrations of the test material were prepared and each concentration was injected intradermally into each intradermal range-finding animal using a syringe attached to a hypodermic needle at: 0.1, 1.0, 3.0 and 5.0 %.
- The test sites of the intradermal range-finding animals were graded for irritation at approximately 24 and 48 hours following intradermal injections using the Dermal Grading System.


MAIN SENSITISATION STUDY

INTRADERMAL INDUCTION
- On the day prior to induction (day -1 ), all test, DNCB test, control and DNCB control animals were weighed and the hair was removed from the scapular area of the animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
- On the day following clipping (day 0), three pairs of intradermal injections were made in the clipped area of all sensitization study animals. The injections were kept within an approximate 2 x 4 cm area with one row of three injections on each side of the back bone.
- Injections for the test animals were as indicated below:
Injection Pair A: 0.1 mL of FCA emulsion
Injection Pair B: 0.1 mL of 5.0 % w/v test material/PEG 400
Injection Pair C: 0.1 mL of 5.0 % w/v test material/FCA emulsion
- Injections for the challenge and re-challenge control animals were as indicated below:
Injection Pair A: 0.1 mL of FCA emulsion
Injection Pair B: 0.1 mL of PEG 400
Injection Pair C: 0.1 mL of 5.0 % w/v PEG 400/FCA emulsion
- Injections for the DNCB test animals were as indicated below:
Injection Pair A: 0.1 mL of FCA emulsion
Injection Pair B: 0.1 mL of DNCB preparation
Injection Pair C: 0.1 mL of DNCB/FCA emulsion
- Injections for the DNCB control animals were as indicated below:
Injection Pair A: 0.1 mL of FCA emulsion
Injection Pair B: 0.1 mL of acetone/propylene glycol
Injection Pair C: 0.1 mL of acetone/propylene glycol/FCA emulsion

TOPICAL INDUCTION
- On the day prior to topical induction (day 6), the guinea pigs had the hair removed with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures. Following clipping, 0.5 mL of 10 % w/w sodium lauryl sulphate in petrolatum was spread over the intradermal injection sites of all study animals.
- On study day 7, any residual sodium lauryl sulphate preparation was removed with dry gauze and 0.8 mL of the appropriate material was prepared and applied to the animals:
Test group: 10 % test material
Challenge control: 100 % PEG 400
Re-challenge control: 100 % PEG 400
DNCB Test group: 0.1 % DNCB in Acetone/Propylene glycol
DNCB Control: 0.1 % Acetone/Propylene glycol 0.1 %
- The patch was applied over the intradermal injection sites. The trunk of each animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the patch and the animal was returned to its cage. Approximately 48 hours after dosing, the elastic wrap, tape and patch were removed. The test sites were wiped with gauze moistened in deionised water to remove test material residue and the animals were returned to their cages.

CHALLENGE
- On the day prior to challenge dose administration, the hair was removed from the right side of the test, challenge control, DNCB test and DNCB control animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
- On the following day (day 21), 0.3 mL of the appropriate concentration of the test material and the DNCB positive control were prepared and each was applied to the appropriate animals:
Test group: 1.0 % test material
Challenge control: 1.0 % test material
DNCB Test group: 0.1 % DNCB in Acetone/Propylene glycol
DNCB Control: 0.1 % DNCB in Acetone/Propylene glycol
- The trunk of each animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the chamber and the animal was returned to its cage. Approximately 24 hours after dosing, the elastic wrap, tape and chamber were removed. The test sites were wiped with gauze moistened with deionized water followed by dry gauze to remove test material residue. The animals were then returned to their cages.

RE-CHALLENGE
- On the day prior to re-challenge dose administration, the hair was removed from the left side of the test and re-challenge control animals with a small animal clipper. Care was taken to avoid abrading the skin during the clipping procedures.
- On the following day (day 28), 0.3mL of the appropriate concentration of the test material was prepared and applied to the animals:
Test group: 0.5 or 1.0 % test material
Re-challenge control: 0.5 or 1.0 % test material
The trunk of each animal was wrapped with elastic wrap which was secured with adhesive tape to prevent removal of the chamber and the animal was returned to its cage.
- Approximately 24 hours after dosing, the elastic wrap, tape and chamber were removed. The test sites were wiped with gauze moistened with deionized water followed by dry gauze to remove test material residue. The animals were then returned to their cages.
- The test sites at challenge and re-challenge were graded for dermal irritation at approximately 24 and 48 hours following chamber removal using the Dermal Grading System.

ANALYSIS OF DATA
- The sensitization potential of the test material was based on the dermal responses of the test and control animals at challenge, re-challenge and second re-challenge. Generally, dermal scores ≥ 1 in the test animals with scores of 0 to ± (slight patchy erythema) noted in the controls are considered indicative of sensitisation. Dermal scores of 1 in both the test and control animals are generally considered equivocal unless a higher dermal response (≥ grade 2) is noted in the test animals. Group mean dermal scores were calculated for challenge and re-challenge. A response of at least 30 percent in an adjuvant test was expected for a mild to moderate sensitiser.
Challenge controls:
The challenge and re-challenge controls were dosed with the test material at 0.5 or 1.0 % in PEG 400.
Positive control substance(s):
yes
Remarks:
1-Chloro-2, 4-Dinitrobenzene (DNCB)
Positive control results:
Following challenge with DNCB, all DNCB test animals were noted to have a substantially stronger dermal response than was observed in the corresponding
DNCB control animals. Group mean dermal scores were also noted to be higher in the DNCB test animals as compared to those of the DNCB control animals
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
1.0 %
No. with + reactions:
1
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
1.0 %
No. with + reactions:
1
Total no. in group:
20
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
1.0 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
1.0 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
0.5 %
No. with + reactions:
1
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
0.5 %
No. with + reactions:
1
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
0.5 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
0.5 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
1.0 %
No. with + reactions:
3
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
1.0 %
No. with + reactions:
2
Total no. in group:
20
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
1.0 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
1.0 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.1 %
No. with + reactions:
10
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.1 %
No. with + reactions:
10
Total no. in group:
10

RANGE-FINDING STUDY

- Topical Range-Finding Study: The results of the range-finding study indicated that a test article concentration of 10 % w/v in PEG 400 appropriate for topical induction since it produced a mild to

moderate irritation. A 1.0 % w/v in PEG 400 was chosen for challenge since it was the highest non-irritating concentration.

- lntradermal Range-Finding Study: The results of the range-finding study indicated that a test article concentration of 5.0 % w/v in PEG 400 was appropriate for intradermal induction.

 

 

SENSITISATION STUDY

- Following challenge with 1 0 % w/v test material in PEG 400, dermal scores of 1 were noted in 1/20 test animals at the 24-hour and 48-hour scoring intervals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to

be similar in the test animals as compared to the challenge control animals.

- Following re-challenge with 1.0 % w/v test material in PEG 400, dermal scores of 1 were noted in 3/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 were noted in 2/20 test animals. Dermal reactions in the remaining test and re-challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be similar in the test animals as compared to the re-challenge control animals.

- Following re-challenge with 0.5 % w/v test material in PEG 400, dermal scores of 1 were noted in 1 /20 test animals at the 24-hour and 48-hour scoring intervals. Dermal reactions in the remaining test and re-challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be similar in the test animals as compared to the re-challenge control animals.

- The sensitisation study animals generally gained weight during the test period. The animals appeared in good health.

Interpretation of results:
other: Not sensitising in accordance with EU criteria
Conclusions:
Under the conditions of the study, the test material is not considered to be a skin sensitiser.
Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 406, OPPTS 870.2600 and JMAFF 59 NohSan No 4200, under GLP conditions.

The dermal sensitization potential of the test material, was evaluated in Hartley derived albino guinea pigs. Ten male and ten female guinea pigs received intradermal injections of 5.0 % w/v test material in polyethylene glycol 400 (PEG 400) along with injections of FCA and 5.0 % w/v test material in FCA. One week later, the test animals received a topical application of 10 % w/v test material in PEG 400. Challenge and re-challenge control animals received similar intradermal

and topical treatments except PEG 400 was used in place of the test material. Following a two-week rest period, a challenge was performed whereby the twenty test and ten previously untreated (naive) challenge control guinea pigs were topically treated with 1.0 % w/v test material in PEG 400. Challenge responses in the test animals were compared with those of the challenge control animals. Following a seven-day rest period, a re-challenge was performed whereby the ten test and ten previously untreated (naive) re-challenge control guinea pigs were topically treated with 0.5 % and 1.0 % w/v test material in PEG 400. Re-challenge responses in the test animals were compared with those of the re-challenge control animals.

A positive control group consisting of ten DNCB test and ten DNCB control animals were induced and challenged in the same manner as the test and control animals mentioned above except that 0.1 % w/v DNCB in acetone/propylene glycol and acetone/propylene glycol, alone, were utilised in conjunction with the Freunds preparation for intradermal induction and, 0.1 % w/v DNCB in acetone/propylene glycol was used for topical induction and challenge.

Following challenge with 1.0 % w/v test material in PEG 400, dermal scores of 1 were noted in 1/20 test animals at the 24-hour and 48-hour scoring intervals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be similar in the test animals as compared to the challenge control animals. Following re-challenge with 1.0 % w/v test material in PEG 400, dermal scores of 1 were noted in 3/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 were noted in 2/20 test animals. Dermal reactions in the remaining test and re-challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be similar in the test animals as compared to 'the re-challenge control animals. Following re-challenge with 0.5 % w/v test material in PEG 400, dermal scores of 1 were noted in 1/20 test animals at the 24-hour and 48-hour scoring intervals. Dermal reactions in the remaining test and re-challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be similar in the test animals as compared to the re-challenge control animals.

Following challenge with the positive control DNCB, all DNCB test animals were noted to have a substantially stronger dermal response than was observed in the corresponding DNCB control animals. Group mean dermal scores were also noted to be higher in the DNCB test animals as compared to those of the DNCB control animals. The results of the DNCB positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitisers.

Under the conditions of the study, the test material is not considered to be a skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 406, OPPTS 870.2600 and JMAFF 59 NohSan No 4200, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The dermal sensitization potential of the test material, was evaluated in Hartley derived albino guinea pigs. Ten male and ten female guinea pigs received intradermal injections of 5.0 % w/v test material in polyethylene glycol 400 (PEG 400) along with injections of FCA and 5.0 % w/v test material in FCA. One week later, the test animals received a topical application of 10 % w/v test material in PEG 400. Challenge and re-challenge control animals received similar intradermal

and topical treatments except PEG 400 was used in place of the test material. Following a two-week rest period, a challenge was performed whereby the twenty test and ten previously untreated (naive) challenge control guinea pigs were topically treated with 1.0 % w/v test material in PEG 400. Challenge responses in the test animals were compared with those of the challenge control animals. Following a seven-day rest period, a re-challenge was performed whereby the ten test and ten previously untreated (naive) re-challenge control guinea pigs were topically treated with 0.5 % and 1.0 % w/v test material in PEG 400. Re-challenge responses in the test animals were compared with those of the re-challenge control animals.

A positive control group consisting of ten DNCB test and ten DNCB control animals were induced and challenged in the same manner as the test and control animals mentioned above except that 0.1 % w/v DNCB in acetone/propylene glycol and acetone/propylene glycol, alone, were utilised in conjunction with the Freunds preparation for intradermal induction and, 0.1 % w/v DNCB in acetone/propylene glycol was used for topical induction and challenge.

Following challenge with 1.0 % w/v test material in PEG 400, dermal scores of 1 were noted in 1/20 test animals at the 24-hour and 48-hour scoring intervals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be similar in the test animals as compared to the challenge control animals. Following re-challenge with 1.0 % w/v test material in PEG 400, dermal scores of 1 were noted in 3/20 test animals at the 24-hour scoring interval. At the 48-hour scoring interval, dermal scores of 1 were noted in 2/20 test animals. Dermal reactions in the remaining test and re-challenge control animals were limited to scores of 0 to ±. Group mean dermal scores were noted to be similar in the test animals as compared to 'the re-challenge control animals. Following re-challenge with 0.5 % w/v test material in PEG 400, dermal scores of 1 were noted in 1/20 test animals at the 24-hour and 48-hour scoring intervals. Dermal reactions in the remaining test and re-challenge control animals were limited to scores of

0 to ±. Group mean dermal scores were noted to be similar in the test animals as compared to the re-challenge control animals.

Following challenge with the positive control DNCB, all DNCB test animals were noted to have a

substantially stronger dermal response than was observed in the corresponding DNCB control animals. Group mean dermal scores were also noted to be higher in the DNCB test animals as compared to those of the DNCB control animals. The results of the DNCB positive control study

demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitisers.

Under the conditions of the study, the test material is not considered to be a skin sensitiser.


Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin sensitisation.