Registration Dossier

Administrative data

Description of key information

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:
disregarded due to major methodological deficiencies
Study period:
Jul/Aug 1994
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study.
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
Buehler test
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc.
- Weight at study initiation: 413 to 688 g
- Housing: individually housed in wire mesh suspension cages
- Diet: Teklad Guinea Pig Diet ad libitum
- Water: ad libitum
- Acclimation period: at least 4 days

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES:
1994-07-19 - 1994-08-31
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
The test substance was used as supplied.
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
The test substance was used as supplied.
No. of animals per dose:
10 males and 10 females for induction and 5 males and 5 females at challenge.
Details on study design:
RANGE FINDING TESTS: An irritation screening test was conducted prior to induction.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6h once per week for 3 weeks
- Test groups: 10 males and 10 females
- Control group: No
- Site: A clipped area on the back of each animal (site 1) was used for all induction exposures
- Frequency of applications: once per week
- Duration: 6 h once per week for 3 weeks
- Concentrations: test material applied as received i.e. undiluted

B. PRIMARY CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 2 weeks after the last induction exsposure
- Exposure period: 6 h
- Test groups: 10 males and 10 females previously induced
- Control group: 5 males and 5 females naive i.e. not previously induced
- Site: Two clipped areas on the back of each animal (sites 2 and 5) were used for the primary challenge exposure.
- Concentrations: test material applied as received i.e. undiluted
- Evaluation (hr after challenge): 24 and 48 h

C. RECHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 1 week after the primary challenge exsposure
- Exposure period: 6 h
- Test groups: 10 males and 10 females previously induced and challenged
- Control group: 5 males and 5 females naive i.e. not previously induced
- Site: Two clipped areas on the back of each animal (sites 4 and 5) were used for the rechallenge exposure
- Concentrations: test material applied as received i.e. undiluted
- Evaluation (hr after challenge): 24 and 48 h
Challenge controls:
No details available.
Positive control substance(s):
yes
Remarks:
α-hexylcinnamaldehyde
Positive control results:
Historical positive control data are maintained by Hill Top Labs, the positive control results confirm the reliability of the testing methods used.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. No with. + reactions: 1.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
No. with + reactions:
2
Total no. in group:
20
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. No with. + reactions: 2.0. Total no. in groups: 20.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
No. with + reactions:
5
Total no. in group:
20
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. No with. + reactions: 5.0. Total no. in groups: 20.0.
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
No. with + reactions:
4
Total no. in group:
20
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. No with. + reactions: 4.0. Total no. in groups: 20.0.
Reading:
1st reading
Hours after challenge:
24
Group:
other: naive animals not previously induced
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group:
Reading:
2nd reading
Hours after challenge:
48
Group:
other: naive
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group:
Reading:
rechallenge
Hours after challenge:
24
Group:
other: naive
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group:
Reading:
rechallenge
Hours after challenge:
48
Group:
other: naive
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group:

The incidence and severity of responses were calculated as follows:

 

 

Group

Incidence of Responses

Mean Severity Scores

24h

48h

0

±

1

2

3

0

±

1

2

3

24 h

48 h

Primary Challenge

Test

3

16

1

0

0

4

14

2

0

0

0.5

0.5

Naïve

3

7

0

0

0

3

7

0

0

0

0.4

0.4

Rechallenge

Test

0

15

3

2

0

1

15

1

3

0

0.7

0.7

Naive

0

9

1

0

0

1

9

0

0

0

0.6

0.5

Interpretation

Grades of 1 or greater in an induced group generally indicate sensitisation, provided grades of less than 1 are seen on respective control animals. If grades of 1 or greater are noted on control animals, then the reactions of respective test animals which exceed the most severe control reaction are presumed to be due to sensitisation.

The results of the primary challenge and rechallenge expressed in terms of both incidence and severity of responses and an assessment by comparison of responses in the test groups to those of the corresponding controls;

1. Incidence - the number of animals in each group showing a specific response at either the first or second readings divided by the total number of animals tested in that group (e.g. 10/20);

2. Severity - the sum of the test grades divided by the total number of animals tested in a given group determined separately for both the first and second readings (e.g. 0.8 - 0.7). Grades of ± are equal to 0.5 for the purpose of calculating severity indices. All average grades are to be rounded to the nearest tenth of a unit, unless the severity is between 0 and 0.05, in which case the severity is rounded to the nearest hundredth of a unit.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test report describes a GLP guideline study. The undiluted test substance is considered to be a sensitiser.
Executive summary:

A skin sensitisation study was conducted using a similar procedure to that described in the current OECD Guideline 406 (Morris, 1994). The test material (CAS No. 50530-43-3) was applied at a volume of 0.3 mL to the backs of 10 male and 10 female Guinea Pigs. The test article was left in contact with the skin for 6 hours. This prodedure was carried out once per week for 3 weeks (induction phase). A primary challenge was then carried out 2 weeks later using the same application procedure as used in the induction phase. This procedure was carried out in the induced animals and in a further group of naive animals. A rechallenge application was then carried out one week after the primary challenge. Scoring was conducted at 24 and 48 h following the primary challenge and the rechallenge phases. The incidence of grade 1 responses in the test group (2 of 20) was compared to that of the naive control group (0 of 10). The incidence and severity of these responses in the test group were greater than those produced by the naive control group indicating that sensitisation to the test material had been induced. The incidence of grade 2 responses in the test group (3 of 20) was compared to that of the naive control group (0 of 10). The incidence and severity of these responses in the test group were again greater than those produced by the naive control group confirming that sensitisation to the test material had been induced.

Under EEC Guidelines, grades of 1 or greater in ≥ 15 % of the test animals results in the classification of the test material as a sensitiser for European labelling purposes. Therefore in accordance with EEC Guidelines, the test material would be labelled as a sensitiser based on the results of the rechallenge exposure.

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

Skin sensitisation

There are two key studies available in order to evaluate the skin sensitisation potential of the target substance (CAS No. 91648-65-6).

In the first key study, the dermal sensitisation potential of the target substance (CAS No. 91648 -65 -6) was evaluated in Hartley-derived albino guinea pigs following OECD Guideline 406, EPA OPPTS 870.2600 and EU Method B.6 (Rodabaugh, 2006). Ten male and ten female guinea pigs were topically treated with 100 % (as received) test material once per week, for three consecutive weeks. Following a 2-week rest period, a challenge was performed whereby the 20 test and 10 previously untreated (naive) challenge control guinea pigs were topically treated with 100 % (as received) test material. Challenge responses in the test animals were compared with those of the challenge control animals. Anα-Hexylcinnamaldehyde (HCA) positive control group consisting of ten HCA test and ten HCA control guinea pigs was included in this study. The animals were treated as above with the HCA test animals receiving 5 % w/v HCA in ethanol for induction and 2.5 % and 1.0 % w/v HCA in acetone for challenge. Following challenge with 100 % test material, dermal reactions in the test animals were limited to scores of 0 to ±. Dermal reactions in the challenge control animals were limited to scores of 0. Group mean dermal scores were noted to be slightly higher in the test animals as compared with the challenge control animals. Following challenge with 2.5 % w/v HCA in acetone, dermal scores of 1 were noted in 2/10 HCA test animals at the 24- and 48-hour scoring intervals. Dermal reactions in the remaining HCA test animals were limited to scores of 0 and ±. Dermal reactions in the HCA challenge control animals were limited to scores of 0. Group mean dermal scores were noted to be higher in the HCA test animals as compared with the HCA challenge control animals. Following challenge with 1.0 % w/v HCA in acetone, a dermal score of 1 was noted in 1/10 HCA test animals at the 24-hour scoring interval. Dermal reactions in the remaining HCA test animals were limited to scores of 0 and ±. Dermal reactions in the HCA challenge control animals were limited to scores of 0. Group mean dermal scores were noted to be higher in the HCA test animals as compared with the HCA challenge control animals.

In the second skin sensitisation study (including topical range-finding study prior to the induction phase), the test was conducted following EPA OPPTS 870.2600 and OECD Guideline 406 (Smedley, 2003). The target substance (CAS No. 91648-65-6) was applied at a volume of 0.3 mL to the backs of 10 male and 10 female guinea pigs. Test concentrations were selected as followed: 100 % during the induction phase, 75 % during the challenge phase (on induced and non-induced animals), 75 % and 50 % during the rechallenge phase (on induced and non-induced animals). On the day prior to test material exposure, the hair was removed from each of the animals’ backs. The test preparations were applied into 25 mm test chambers and then applied to the clipped surface as quickly as possible. The chambers were occluded with elastic wrap which was secured with adhesive tape to prevent removal of the chamber. Approximately six hours later the binding materials were removed. The test sites were wiped with gauze moistened in mineral oil, followed by dry gauze, followed by gauze moistened in deionized water, followed by dry gauze, to remove test article residue.This procedure was carried out once per week for 3 weeks (induction phase). A primary challenge was then carried out 2 weeks later using the same application procedure as used in the induction phase. This procedure was carried out in the induced animals and in a further group of naive animals. A rechallenge application was then carried out one week after the primary challenge. Scoring was conducted at 24 h (1st reading) and 48 h (2nd reading) following the primary challenge and the rechallenge phases. 2 animals showed positive skin reactions in the test group (75 % substance concentration) at the 2nd reading. The same observation was recorded at the first and second readings of the rechallenge in the test group with 50 % and 75 % test concentrations, respectively. One female had transient incidences of entire body reddened and warm to the touch (days 8 to 12), faeces small in size and decreased food consumption (day 8). Since these findings were transient in nature and limited to just one animal, these findings are not considered to be test article related. The sensitisation study animals gained weight during the test period and generally appeared in good health. Hexylcinnamaldehyde (HCA) was used as positive control. Following challenge with this substance, HCA test animals were noted to have a stronger dermal response than was observed in the corresponding HCA control animals (4 out of 10 animals showed a positive response at 48 h at the 2.5 % dose level). The results of the HCA positive control study demonstrated that a valid test was performed and indicated that the test design would detect potential contact sensitisers.

Considering to be a supporting study, the read-across substance (1,3,4-Thiadiazolidine-2,5-dithione, reaction products with hydrogen peroxide and tert-dodecanethiol) was tested in a GLP study according to OECD Guideline 406 for its sensitising potential in order to determine whether there is any specific (sensitising) or non-specific (irritant) stimulating potential of the test substance (Grunert, 1993). In a pilot study the test item was tested dermally, occlusive and thinned to 12.5 %, 25 %, 50 % and 100 % in sesame oil over a period of 6h with a single exposure. Each dose group consisted of 2 guinea pigs (Pirbright White, age < 1a), an area of 4 cm x 6 cm was clipped and exposed to 0.3 mL of the test item, the post-exposure period was 72 h. Additionally, a control group was not exposed to the test item. After 1 h, 1 d, 2 d and 3 d the treated skin was checked for irritation. At no time point and in no animal any skin reactions were observed. Therefore, the undiluted test item was chosen for the main study. In the main study, a test group and a control group consisting each of 10 guinea pigs (5 males / 5 females) were used. The test group was exposed occlusive during the induction phase three times (day 0, 7, 14) over 6 h to 0.3 ml the test item onto 4 cm x 6 cm clipped skin. The control group was not exposed to the test item but was treated the same way regarding clipping and the occlusive bandage. After two weeks free from all treatment the test item was applied similar to the induction phase on day 28 to both groups (test animals as well as control animals) over 6 h. 24 h and 48 h after the challenge skin readings were performed. No animal, neither control nor test group, exhibited any skin reaction.

In another supporting study, the skin sensitisation study was conducted using a similar procedure to that described in the current OECD Guideline 406 (Morris, 1994). The read-across substance (CAS No. 50530-43-3), 5 % dilution in base oil (EX-80/90 BO), was applied at a volume of 0.3 mL to the backs of 10 male and 10 female guinea pigs. The test article was left in contact with the skin for 6 hours. This procedure was carried out once per week for 3 weeks (induction phase). A primary challenge was then carried out 2 weeks later using the same application procedure as used in the induction phase. This procedure was carried out in the induced animals and in a further group of naive animals. A rechallenge application was then carried out one week after the primary challenge. Scoring was conducted at 24 and 48 h following the primary challenge and the rechallenge phases. Following primary challenge with the test material, as received, the incidence of grade 1 responses in the test group (4 of 20) was compared to that of the naive control group (3 of 10). The incidence and severity of these responses in the test group were essentially comparable to those produced by the naive control group indicating that sensitisation to the test material had not been induced. Following concurrent primary challenge using the vehicle EX-80/90 BO (base oil), as received, the incidence of grade 1 responses in the test group (6 of 20) was compared to that of the naive control group (3 of 10). The incidence and severity of these responses in the test group were essentially comparable to those produced by the naive control group indicating that sensitisation to EX-80/90 BO (base oil) had not been induced.

In a disregarded study, the skin sensitisation test was conducted using a similar procedure to that described in the current OECD Guideline 406 (Morris, 1994). The read-across substance (CAS No. 50530-43-3) was applied at a volume of 0.3 mL to the backs of 10 male and 10 female Guinea Pigs. The test article was left in contact with the skin for 6 hours. This prodedure was carried out once per week for 3 weeks (induction phase). A primary challenge was then carried out 2 weeks later using the same application procedure as used in the induction phase. This procedure was carried out in the induced animals and in a further group of naive animals. A rechallenge application was then carried out one week after the primary challenge. Scoring was conducted at 24 and 48 h following the primary challenge and the rechallenge phases. The incidence of grade 1 responses in the test group (2 of 20) was compared to that of the naive control group (0 of 10). The incidence and severity of these responses in the test group were greater than those produced by the naive control group indicating that sensitisation to the test material had been induced. The incidence of grade 2 responses in the test group (3 of 20) was compared to that of the naive control group (0 of 10). The incidence and severity of these responses in the test group were again greater than those produced by the naive control group confirming that sensitisation to the test material had been induced.

Justification: This study is considered as disregarded which is mainly based on the chemical structure of the test substance. In general, the sensitisation potential of a substance is strongly influenced by its molecular size and its lipophilicity. Due to the fact, that the read-across substance (CAS No. 50530-43-3) is less lipophilic than the target substance (CAS No. 91648 -65 -6) a sensitisation potential for the read-across substance is more likely. In conclusion, the ambivalent results can be explained by these substance properties and the study conducted by Morris (1994) is thus considered as disregarded.


Migrated from Short description of key information:
Only slight effects were observed in the key study following OECD Guideline 406, EPA OPPTS 870.2600 and EU Method B.6 (Rodabaugh, 2006). In the second key study following EPA OPPTS 870.2600 and OECD Guideline 406 (Smedley, 2003), similar effects were reported.

Justification for selection of skin sensitisation endpoint:
There are two key studies available which have been conducted using the target substance.

Justification for classification or non-classification

Skin sensitisation

According to the classification criteria outlined in section 3.4.2.2 (Guidance on the Application of CLP criteria, 2012), if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or if there are positive results from an appropriate animal test, the classification as Skin Sensitiser is assigned.

Referring to non human data, significant skin sensitising effects are described if redness occured in ≥ 15 % of the test animals (Buehler occluded patch test). Based on the available key information, this substance does not meet the requirement under EU CLP (Regulation (EC) No. 1272/2008) for classification as a skin sensitiser.The substance is not sensitising.