long chain alkyl thio carbamide metal complex

Endpoint summary

Description of key information

The substance was tested in a Guinea-Pig Maximisation Test, which employs intradermal injection and use of adjuvant. Upon challenge with 0.5% test substance (the highest non-irritating concentration), six of the 20 animals treated with the test substance at induction showed Grade 1 erythema at the 24-hour observation, and four animals showed Grade 1 erythema at the 48-hour observation. The control animals showed no response to challenge. Upon rechallenge with 0.2% test substance, no responses were seen in either treatment or control animals. The substance was concluded to be a weak sensitiser in this test.

A read-across GPMT study has been added to support the chemical and toxicological similarities between the registration substance (EC: 457 -320 -2) and EC: 434 -650 -5. EC: 434 -650 -5 also causes skin sensitisation with 60% treated animals during the challenge and 30% of animals during the rechallenge exhibiting a reaction that could be attributed to an allergic response.

Short description of key information:
The substance was concluded to be a weak sensitiser in a guinea-pig maximisation test (category 1B).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 May 2002-13 June 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

not specified

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

This study was conducted before the LLNA was adopted in 2002.

Species:

guinea pig

Strain:

other: Hartley-derived albino

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Hilltop Lab Animals, Inc., Scottdale, PA
- Age at study initiation: males approximately 7 weeks; females approximately 9 weeks
- Weight at study initiation: males 396-483 g; females 393-458 g
- Housing: Individually in suspended stainless steel cages
- Diet (e.g. ad libitum): PMI Certified Guinea Pig Chow ~5026 (purina Mills, Inc.), ad libitum
- Water (e.g. ad libitum): municipal tap water treated by reverse osmosis, ad libitum
- Acclimation period: 5 days minimum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 36-77
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal and epicutaneous

Vehicle:

other: mineral oil

Concentration / amount:

Concentration of test material used at induction: Intradermal injection: 5% w/w in mineral oil
and 5% w/w in Freund's complete adjuvant emulsion
Epicutaneous application: 25% in mineral oil
Concentration of test material and vehicle used for each challenge: Challenge application: 0.5% and 0.2% w/w in mineral oil.

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

Challenge application: 0.5% w/w in mineral oil.

Adequacy of challenge:

highest non-irritant concentration

Route:

epicutaneous, occlusive

Vehicle:

other: mineral oil

Concentration / amount:

0.2% w/w in mineral oil

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Number of animals in test group: 10/sex
Number of animals in negative control group: 5/sex

Details on study design:

RANGE FINDING TESTS: Intradermal and topical range-finding studies were carried out in order to determine the concentrations to be used in the main studies.

MAIN STUDY
A. INDUCTION EXPOSURE - INTRADERMAL AND EPICUTANEOUS INDUCTION
- No. of exposures: 2 (1 intradermal and 1 epicutaneous)
- Exposure period: indefinite (intradermal); 48 hours (epicutaneous, occlusive cover)
- Test groups: 1
- Control group: yes
- Site: back
- Frequency of applications: single set of 6 injections (3 pairs) at day 0; single epicutaneous administration at day 8
- Duration: not applicable
- Concentrations: Injections 5% test substance in mineral oil or Freund's complete adjuvant emulsion; epicutaneous 25% test substance in mineral oil

B. CHALLENGE EXPOSURE
- No. of exposures: 1 upon challenge and 1 upon rechallenge
- Day(s) of challenge: day 21 and day 28
- Exposure period: approximately 24 hours
- Test groups: 1
- Control group: yes
- Site: back
- Concentrations: 0.5% in mineral oil (challenge) and 0.2% in mineral oil (rechallenge)
- Evaluation (hr after challenge/rechallenge): approximately 24 and 48

Challenge controls:

5/sex challenged with 0.5% in mineral oil; another group of 5/sex rechallenged with 0.2% in mineral oil (volume 0.3 ml in each case; occlusive cover). Evaluation after approximately 24 hours in each case.

Positive control substance(s):

yes

Remarks:

alpha-Hexylcinnamaldehyde; test not carried out concurrently with present study, but within 6 months. Induction at 5% in propylene glycol and challenge at 0.5% and 1% in propylene glycol.

Positive control results:

A contact sensitization response was observed.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.5 %

No. with + reactions:

6

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: .5 %. No with. + reactions: 6.0. Total no. in groups: 20.0.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.2 %

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: .2 %. No with. + reactions: 0.0. Total no. in groups: 20.0.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.5 %

No. with + reactions:

4

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: .5 %. No with. + reactions: 4.0. Total no. in groups: 20.0.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0.2 %

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: .2 %. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.5 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: .5 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.2 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: .2 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.5 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: .5 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0.2 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: .2 %. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

1% w/v HCA in propylene glycol

No. with + reactions:

17

Total no. in group:

20

Clinical observations:

edema, eschar, desquamation and/or superficial lightening

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

1% HCA in propylene glycol

No. with + reactions:

16

Total no. in group:

20

Clinical observations:

edema, eschar, desquamation and/or superficial lightening

Remarks on result:

positive indication of skin sensitisation

Reading:

rechallenge

Hours after challenge:

24

Group:

positive control

Dose level:

0.5% HCA in propylene glycol

No. with + reactions:

11

Total no. in group:

20

Clinical observations:

edema

Remarks on result:

positive indication of skin sensitisation

Reading:

rechallenge

Hours after challenge:

48

Group:

positive control

Dose level:

0.5% HCA in propylene glycol

No. with + reactions:

11

Total no. in group:

20

Clinical observations:

edema and/or desquamation

Remarks on result:

positive indication of skin sensitisation

Maximum concentration not causing irritating effects in range finding intradermal injection test was 5% w/w in mineral oil. This was the highest concentration indicated under guidelines for the test; it produced no systemic toxicity and only localized reactions at the injection sites. In the range-finding epicutaneous study, concentrations of 1, 2.5, 5, 10, 25, 50, 75 and 100% were assessed. A concentration of 25% produced a mild to moderate [irritation] response; 0.5% apparently produced minimal irritation and was considered to be the "highest nonirritating concentration"; it was used as a challenge concentration.

 

Evidence of sensitization of each challenge concentration: Following challenge with 0.5% w/w test substance in mineral oil, dermal scores of 1 were noted in 6/20 animals at the 24-hour scoring interval. At the 48 hour scoring interval, dermal scores of 1 were noted in 4/20 animals. Dermal reactions in the remaining test and challenge control animals were limited to scores of 0 to +/- ("slight patchy erythema"; note this was seen in all of the challenge control animals at 24 hours). Group mean dermal scores were noted to be higher in the test animals as compared to the challenge control animals.

 

Following rechallenge with 0.2% w/w test substance in mineral oil, dermal reactions were limited to scores of 0 to +/- in all test and rechallenge control animals following the 24 and 48 hour scoring intervals.

 

Other observations: One rechallenge control animal was found dead on Day 9, but as the animal had been dosed with vehicle the death was not considered to be test-article related.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

In a GLP study conducted according to EPA OPPTS 870.2600, the substance was considered to be a weak skin sensitiser in a guinea pig maximisation test

Executive summary:

In a GLP study conducted according to EPA OPPTS 870.2600, the substance was tested for dermal sensitisation potential in a guinea pig maximisation test. Briefly, animals (10/sex) were induced initially with intradermal injections of 5% in mineral oil and 5% in Freund's complete adjuvant emulsion and then, one week later, with topical administration of a concentration of 25% in mineral oil (under occlusive cover for 48 hours). After a rest period of two weeks, animals were challenged with topical administration of 0.5% in mineral oil. They were rechallenged after a further week with a concentration of 0.2% in mineral oil.

 

Upon challenge with 0.5% in mineral oil, 6/20 animals had dermal scores of 1 (slight, but confluent or moderate patchy erythema) after 24 hours, and only 4 animals retained this degree of redness after 48 hours. Dermal reactions in the remaining test and challenge control animals were limited to no or only "slight patchy erythema". There were no significant differences in reactions between treated and control animals upon rechallenge with 0.2% in mineral oil (at most only "slight patchy erythema" was seen, and this occurred in both test and rechallenge control animals).

 

In conclusion, the substance can be considered to be a weak skin sensitiser when tested in a guinea pig maximisation test.

Reference 2

Endpoint:

skin sensitisation: in chemico

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Reference 3

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

October 1999 - December 1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study was conducted before OECD 429 TG was adopted in 2010.

Specific details on test material used for the study:

- Source and lot/batch number of test material: 6786-35
- Expiration date of the lot/batch: August 2004
- Purity: Considered pure
- Storage condition of test material: Room temperature

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

- Source: Charles River Laboratories Kingston Facility, NY
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 5 weeks
- Weight at study initiation: 323 to 381 grams
- Housing: Single housed in suspended stainless steel and wire mesh caging with absorbant paper below cages
- Diet: PMI feeds ad libitum - no known contaminants in the feed
- Water: ad libitum - no known contaminants in the water
- Temperature (°F): 64 to 72
- Humidity (%): 30-70%
- Photoperiod: approximately 12 hours light and 12 hours dark

Route:

intradermal

Vehicle:

peanut oil

Concentration / amount:

0.1 ml - 5% test substance in peanut oil

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:

0.5 ml - 100% test substance

Day(s)/duration:

48 hours

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

0.4 ml - 100% test substance

Day(s)/duration:

24 hours

Adequacy of challenge:

highest non-irritant concentration

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

olive oil

Concentration / amount:

0.4ml - 50% test substance in olive oil

Day(s)/duration:

24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

20

Details on study design:

Intradermal Induction (Day 0)
Preparation of Animals:
An area near the scapula in the mid-dorsal region of all animals was clipped on the day prior to intradermal injection of the test substance and/or carrier. All animals were clipped using Oster A-5 Small Animal Clippers equipped with size 40 blades.
Administration of Test Substance:
A pair of 0.1 ml injections of the following mixtures were administered intradermally to each of three sites in the mid-dorsal region of all animals, near the scapula:
Site 1 - diluted FCA to treated and irritation control group;
Site 2 - test substance in peanut oil to the treated group, undiluted peanut oil to the irritation control group;
Site 3 - test substance in diluted FCA to the treated group (concentration was adjusted so that the absolute amount of substance was the same as that administered at Site 2); peanut oil in diluted FCA to the irritation control group.
The solutions were injected intradermally with an appropriate size syringe and 25 gauge needle. A row of three injections, 0.1 ml of each solution, was given along both sides of the dorsal midline for a total of six injections. Solutions for Site 1 and Site 2 were injected close together and near the first thoracic vertebra; the solution for Site 3 was injected more caudally. All injections were made 1-2 cm from the midline and within the boundaries of the clipped region.

Induction by Occlusive Topical Application (Day 7):
Preparation of Animals (Day 6)
The fur in the previously treated scapular area was reclipped on the day prior to topical application of the test substance or carrier.
Administration of Test Substance
Seven days after intradermal induction, 0.5 ml of 100% (neat) test substance was applied topically over the previously treated and clipped scapular area of the treated group animals. The irritation control group animals received 0.5 ml of carrier (100% peanut oil). The test substance and/or carrier was applied to the skin under a 2 x 4 cm filter paper, covered by overlapping plastic tape (Dermicel) and firmly secured to the torso of the animals with elastic adhesive bandaging (Elastikon). The pads and sleeving were removed from both the treated and control group animals after approximately 48 hours. The skin was gently wiped free of remaining test substance and/or carrier with dry gauze.

Challenge by Occlusive Topical Application (Day 21)
Preparation of Animals
The fur in an area on the left and right flank in the abdominal region was clipped on the day prior to challenge dosing of the test substance.
Administration of Test Substance
Twenty-one days after intradermal induction, 0.4 ml of 100% (neat) test substance was applied topically to the clipped area on the left flank and 0.4 ml of carrier (olive oil) alone was applied to the right flank of all treated and ten irritation control group animals. The test substance and the carrier each were applied to their respective Hilltop Chamber (Hilltop Research Inc., Cincinnati, Ohio) and firmly secured to the torso of the animals with elastic adhesive bandaging (Elastikon). The chambers and sleeves were removed from both the treated and irritation control animals after approximately 24-hours and gently wiped with paper towels and peanut oil to remove any residual substance.
Approximately 21 hours after removal of the challenge patch, the dose sites were clipped.

Rechallenge by Occlusive Topical Application (Day 28)
Preparation of Animals
The fur in an area on the left and right flank in the abdominal region was clipped on the day prior to rechallenge dosing of the test substance.
Administration of Test Substance
Twenty-eight days after intradermal induction, 0.4 ml of 50% test substance diluted in olive oil was topically applied to the clipped area on the right flank and 0.4 ml of 100% olive oil was applied to the left flank of all treated and ten irritation control group animals. The test substance and the carrier each were applied to their respective Hilltop Chamber (Hilltop Research Inc., Cincinnati, Ohio) and firmly secured to the torso of the animals with elastic adhesive bandaging (Elastikon). The chambers and sleeves were removed from both the treated and irritation control animals after approximately 24-hours and gently wiped with paper towels and olive oil to remove any residual substance.
Approximately 21 hours after removal of the challenge patch, the dose sites were clipped.

The animals were examined for viability twice daily Monday through Friday, and once daily on weekends and/or holidays (if applicable).
Clinical observations were made as to the nature, onset, severity and duration of toxicological signs after dosing on Day 0, and on Days 7, 14, 21, 28, and prior to sacrifice.
Body weights were recorded pretest for sorting purposes, prior to dose initiation on Day 0, and on Days 7, 14, 21, 28, and prior to sacrifice.
Dermal responses were evaluated approximately 1 and 24-hours after intradermal induction using descriptive terminology. Dermal responses were evaluated on Day 9 approximately 1 hour after removal of the topical induction patch and on Day 10 approximately 24-hours after removal of the topical induction patch. Dermal responses were evaluated at approximately 24 and 48 hours after removal of the challenge patch (Days 23 and 24, respectively), and rechallenge patch (Days 30 and 31, respectively). All scoring was made according to the scoring system described in Key A.
After the final dermal observations, all animals were sacrificed by carbon dioxide asphyxiation and discarded without further examination.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100% test substance

No. with + reactions:

12

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100% test substance

No. with + reactions:

13

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

50% test substance in olive oil

No. with + reactions:

6

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

50% test substance in olive oil

No. with + reactions:

3

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100% test substance

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

Edema was not observed in any animal

Remarks on result:

other: Irritation response

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100% test substance

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

Edema was not observed in any animal

Remarks on result:

other: Irritation response

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

50% test substance in olive oil

No. with + reactions:

1

Total no. in group:

10

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

50% test substance in olive oil

No. with + reactions:

1

Total no. in group:

10

The Primary Irritation Test (PIT) was performed to determine the concentration of test substance to be used for intradermal induction. Three concentrations (0.1, 1, and 5%) were administered intradermally to two animals per concentration. The injection sites were evaluated at 24 and 48 hours post-injection. The 0.1 and 1.0% concentrations produced slight to moderate redness while the 5.0% concentration produced slight to extreme redness. Tan centers were noted in the 0.1% treated animals at both evaluations. Tan and brown centers were noted in the 1.0% treated animals at the 24-hour and 48-hour evaluations. Brown centers were noted in the 5.0% treated animals at the 24-hour and 48-hour evaluations. No dose produced eschar or necrosis. Therefore, 5.0% was selected as the concentration for the intradermal induction.

A second PIT was conducted to select doses for topical induction. Four concentrations (5, 25, 50, 100%) were applied topically to five animals. Grade ± or 1 irritation was noted for all concentrations. Therefore the neat test material was selected for the topical induction.

One week prior to the main study challenge, 0.1, 1.0, and 100% were applied topically to five animals to determine the challenge dose. Additionally, the carrier for the topical challenge, olive oil, was applied to the animals.

Challenge Dermal scores:

		0	±	1	2	3	N
TREATED GROUP
24 HOUR	LEFT FLANK 100% TEST SUBSTANCE	0	0	7	9	4	20
	RIGHT FLANK 100% OLIVE OIL	11	7	2	0	0	20
48 HOUR	LEFT FLANK 100% TEST SUBSTANCE	0	0	6	11	3	20
	RIGHT FLANK 100% PEANUT OIL	14	6	0	0	0	20
IRRITATION CONTROL GROUP
24 HOUR	LEFT FLANK 100% TEST SUBSTANCE	0	3	6	1	0	10
	RIGHT FLANK 100% OLIVE OIL	6	3	1	0	0	10
48 HOUR	LEFT FLANK 100% TEST SUBSTANCE	2	1	6	1	0	10
	RIGHT FLANK 100% OLIVE OIL	7	3	0	0	0	10

Rechallenge dermal scores:

		0	±	1	2                           3		N
TREATEDGROUP
24 HOUR	LEFT FLANK 100% OLIVE OIL	16	3	1	0	0	20
	RIGHT FLANK 50% TEST SUBSTANCE IN OLIVE OIL	0	7	7	6	0	20
48 HOUR	LEFT FLANK 100% OLIVE OIL	19	1	0	0	0	20
	RIGHT FLANK 50% TEST SUBSTANCE IN OLIVE OIL	3	9	5	3	0	20
IRRITATION CONTROL GROUP
24 HOUR	LEFT FLANK 100 % OLIVE OIL	10	0	0	0	0	10
	RIGHT FLANK 50 % TEST SUBSTANCE IN OLIVE OIL	7	2	1	0	0	10
48 HOUR	LEFT FLANK 100% OLIVE OIL	10	0	0	0	0	10
	RIGHT FLANK 50% TEST SUBSTANCE IN OLIVE OIL	9	0	1	0	0	10

Challenge:

In the irritation control group, topical application of olive oil produced Grade ± response in three of the ten animals and Grade 1 responsein one animal at the 24-hour observation. At the 48-hour observation, Grade ± response was observed in three animals. Edema was not observed in any irritation control animal at the 24 or 48-hour observations but desquamation was noted in one animal at the 48-hour evaluation.

During the challenge 12 out of 20 of the treated animals (60%) exhibited a reaction that could be attributed to an allergic response. According to the ranking system proposed by Magnusson and Kligman (1969) the test substance would be assigned an all ergenicity rating of Grade III (moderate classification).

Rechallenge:

In an attempt to reduce the dermal irritation observed during challenge, the rechallenge was conducted with a concentration of 50.0% in olive oil. Rechallenge dosing with the test substance produced a Grade ± response in seven animals; a Grade 1 response in seven animals and a Grade 2 response in six animals at the 24-hour observation. At the 48-hour observation a Grade ± response was observed in nine animals, a Grade 1 response was observed in five animals, and a Grade 2 response was observed in three animals. Edema was observed in two animals and desquamationwasobserved in four animals at the 24-hour observation; desquamation was observed in 17 animals at the 48-hour observation, and cracking was observed in two animals at the 48-hour observation.

In the irritation control group, topical application of the test substance produced a Grade ± and a Grade 1 response in two and one of the ten animals, respectively, at the 24 observation. At the 48-hour observation, a Grade 1 response was observedin one animal. Edema was not observed in anyirritationcontrol animal at the 24 or 48-hour observations.

Rechallenge dosing with olive oil in the treated groupproduced a Grade ± and a Grade I response in three and one of the 20 animals, respectively, at the 24-hour observation. At the 48-hour observation, a Grade ± response was observed in one animal. Desquamation was noted in one animal at the 48-hour observation. Edema was not observed in any treated group animals at the 24 or 48-hour observations.

In the irritation control group, topical application of olive oil during the rechallenge produced no signs of irritation at either observation interval.

During the rechallenge 6 out of 20 of the treated animals (30%) exhibited a reaction that could be attributed to an allergic response.

All animals survived until study termination. Emaciation and/or little sign of stool were the most frequently observed clinical signs and were noted in one treated group and two irritation control animals. All animals gained weight over their initial Day 0 values.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

At challenge 12 out of 20 of the treated animals (60%) exhibited a reaction that could be attributed to an allergic response. At rechallenge six out of 20 of the treated animals (30%) exhibited a reaction that could be attributed to an allergic response. According to the ranking system proposed by Magnusson and Kligman (1969) the test substance would be assigned an allergenicity rating of Grade III (moderate classification) for either interval.
In conclusion, the induction concentrations elicited the responses required for the proper conduct of a sensitization study. The challenge responses were considered a mixture of irritation and sensitization responses, but the amount of irritation present did not confound interpretation of the data. Rechallenge with a lower concentration reduced the irritation response to a more appropriate level (as seen in the irritation control group responses) and the results of both the challenge and rechallenge revealed the test substance had moderate sensitization potential (modified from Thomas, 1970).

Executive summary:

The allergic contact sensitization potential of the test substance was evaluated in the guinea pig using the Guinea Pig Maximization Test (Magnusson and Kligman, 1969; OECD, 1992; EC, 1993). Twenty female Hartley albino guinea pigs received intradermal injection and topical exposures to the test substance. An irritation control group of 20 female guinea pigs received the induction carrier, peanut oil, through the induction phases of the study and then exposure to the test substance at challenge (ten animals) or rechallenge (ten animals). The challenge carrier, olive oil also was topically challenged or rechallenged in both the treated and irritation control groups. Evaluation of sensitization potential was based on a comparison of the treated and irritation control group dermal scores. The results of the testing facility's most recent reliability test using the positive control material 2-mercaptobenzothiazole (MIST) are reported.

Primary range-finding tests were conducted to determine appropriate concentrations of the test substance to be used in the three phases of dosing. Based on the results of these tests, concentrations of 5%, 100%, and 100% were chosen for the intradermal induction, topical induction, and topical challenge, respectively. Due to irritation observed during challenge, rechallenge was conducted at 50% test substance in olive oil to reduce the amount of background irritation.

Dermal responses were evaluated approximately 1 and 24-hours after the intradermal induction using descriptive terminology. Dermal responses were evaluated 1 and 24-hours (Days 9 and 10) after removal of the topical induction patch, and 24 and 48 hours (Days 23 and 24) after removal of the topical challenge patch, and 24 and 48 hours (Days 30 and 31) after removal of the rechallenge patch using the scoring system described in Key A. Observations were made as to the nature, onset, severity and duration of toxicological signs after dosing on Day 0 and on Days 7, 14, 21, and 28 and on Day 31. Body weights were recorded once at pretest and on Days 0, 7, 14, 21, 28 and 31. After the final observations and weighing’s, all animals were sacrificed by exsanguination following carbon dioxide asphyxiation and discarded without further examination.

All animals survived until study termination. Emaciation and/or little sign of stool were the most frequently observed clinical signs and were noted in one treated group and two irritation control group animals. All animals gained weight over their initial Day 0 values.

Intradermal induction conducted with 5% test substance did not produce eschar or necrosis. Topical induction with the neat test substance produced Grade ± to 2 responses in all animals.

Topical challenge with 100% test substance elicited responses ranging from Grade 1 to 3 in all treated animals and responses ranging from Grade ± to 2 in all irritation control animals at the 24-hour observation. Edema was noted in four treated animals at the 24-hour observation. At the 48-hour observation, the severity of the responses remained essentially the same for the treated group. The response for the irritation control group decreased slightly at the 48-hour observation and irritation was observed in eight of the ten animals. In the treated group, edema was noted in four animals and cracking was noted in three animals at the 48-hour observation.

In an attempt to reduce the irritation observed at challenge in the irritation control group, the topical rechallenge was conducted with 50% test substance in olive oil. The rechallenge elicited responses ranging from Grade ± to 2 in all treated animals at the 24-hour observation. The rechallenge with 50% test substance in olive oil elicited Grade ± and Grade1responses in two and one of the 10 irritation control animals, respectively,at the 24-hour observation: At the 48- hour observation, Grade ± responses were noted in nine treated animals, Grade 1 responses were noted in five treated animals, and Grade 2 responses in three treated animals. A Grade 1 response was noted in one of 10 irritation control animals at this interval.

Topical application of olive oil at challenge and rechallenge elicited primarily Grade ± responses but occasionally Grade 1 responses were observed. Responses generally decreased by 48 Hours at both challenge and rechallenge.

At challenge 12 out of 20 of the treated animals (60%) exhibited a reaction that could be attributed to an allergic response. At rechallenge six out of 20 of the treated animals (30%) exhibited a reaction that could be attributed to an allergic response. According to the ranking system proposed by Magnusson and Kligman (1969) the test substance would be assigned an allergenicity rating of Grade III (moderate classification)for either interval.

In conclusion, the induction concentrations elicited the responses required for the proper conduct of a sensitization study. The challenge responses were considered a mixture of irritation and sensitization responses, but the amount of irritation present did not confound interpretation of the data. Rechallenge with a lower concentration reduced the irritation response to a more appropriate level (as seen in the irritation control group responses) and the results of both the challenge and rechallenge revealed the test substance had moderate sensitization potential (modified from Thomas, 1970).

Reference 5

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100% test substance

No. with + reactions:

12

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100% test substance

No. with + reactions:

13

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

50% test substance in olive oil

No. with + reactions:

6

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

50% test substance in olive oil

No. with + reactions:

3

Total no. in group:

20

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100% test substance

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

Edema was not observed in any animal

Remarks on result:

other: Irritation response

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100% test substance

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

Edema was not observed in any animal

Remarks on result:

other: Irritation response

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

50% test substance in olive oil

No. with + reactions:

1

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

50% test substance in olive oil

No. with + reactions:

1

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Read-across Challenge Dermal scores:

		0	±	1	2	3	N
TREATED GROUP
24 HOUR	LEFT FLANK 100% TEST SUBSTANCE	0	0	7	9	4	20
	RIGHT FLANK 100% OLIVE OIL	11	7	2	0	0	20
48 HOUR	LEFT FLANK 100% TEST SUBSTANCE	0	0	6	11	3	20
	RIGHT FLANK 100% PEANUT OIL	14	6	0	0	0	20
IRRITATION CONTROL GROUP
24 HOUR	LEFT FLANK 100% TEST SUBSTANCE	0	3	6	1	0	10
	RIGHT FLANK 100% OLIVE OIL	6	3	1	0	0	10
48 HOUR	LEFT FLANK 100% TEST SUBSTANCE	2	1	6	1	0	10
	RIGHT FLANK 100% OLIVE OIL	7	3	0	0	0	10

Challenge:

In the irritation control group, topical application of olive oil produced Grade ± response in three of the ten animals and Grade 1 responsein one animal at the 24-hour observation. At the 48-hour observation, Grade ± response was observed in three animals. Edema was not observed in any irritation control animal at the 24 or 48-hour observations but desquamation was noted in one animal at the 48-hour evaluation.

During the challenge 12 out of 20 of the treated animals (60%) exhibited a reaction that could be attributed to an allergic response.

Read-across Rechallenge dermal scores:

		0	±	1	2                           3		N
TREATEDGROUP
24 HOUR	LEFT FLANK 100% OLIVE OIL	16	3	1	0	0	20
	RIGHT FLANK 50% TEST SUBSTANCE IN OLIVE OIL	0	7	7	6	0	20
48 HOUR	LEFT FLANK 100% OLIVE OIL	19	1	0	0	0	20
	RIGHT FLANK 50% TEST SUBSTANCE IN OLIVE OIL	3	9	5	3	0	20
IRRITATION CONTROL GROUP
24 HOUR	LEFT FLANK 100 % OLIVE OIL	10	0	0	0	0	10
	RIGHT FLANK 50 % TEST SUBSTANCE IN OLIVE OIL	7	2	1	0	0	10
48 HOUR	LEFT FLANK 100% OLIVE OIL	10	0	0	0	0	10
	RIGHT FLANK 50% TEST SUBSTANCE IN OLIVE OIL	9	0	1	0	0	10

Rechallenge:

In an attempt to reduce the dermal irritation observed during challenge, the rechallenge was conducted with a concentration of 50.0% in olive oil. Rechallenge dosing with the test substance produced a Grade ± response in seven animals; a Grade 1response in seven animals and a Grade 2 response in six animals at the 24-hour observation. At the 48-hour observation a Grade ± response was observed in nine animals, a Grade 1response was observed in five animals, and a Grade 2 response was observed in three animals. Edema was observed in two animals and desquamationwasobserved in four animals at the 24-hour observation; desquamation was observed in 17 animals at the 48-hour observation, and cracking was observed in two animals at the 48-hour observation.

In the irritation control group, topical application of the test substance produced aGrade ± and a Grade 1response in two and one of the ten animals, respectively, atthe 24 observation. At the 48-hour observation, a Grade 1 response wasobservedin one animal. Edema was not observed in anyirritationcontrol animal at the 24 or 48-hour observations.

Rechallenge dosing with olive oil inthe treated groupproduced a Grade ± and a Grade I response in three and one of the 20 animals, respectively, at the 24-hour observation. At the 48-hour observation, a Grade ± response was observed in one animal. Desquamation was noted in one animal at the 48-hour observation. Edema was not observed in any treated group animals at the 24 or 48-hour observations.

In the irritation control group, topical application of olive oil during the rechallenge produced no signs of irritation at either observation interval.

During the rechallenge 6 out of 20 of the treated animals (30%) exhibited a reaction that could be attributed to an allergic response.

All animals survived until study termination. Emaciation and/or little sign of stool were the most frequently observed clinical signs and were noted in one treated group and two irritation control animals. All animals gained weight over their initial Day 0 values.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

At challenge 12 out of 20 of the treated animals (60%) exhibited a reaction that could be attributed to an allergic response. At rechallenge six out of 20 of the treated animals (30%) exhibited a reaction that could be attributed to an allergic response. According to the ranking system proposed by Magnusson and Kligman (1969) the test substance would be assigned an allergenicity rating of Grade III (moderate classification) for either interval.
In conclusion, the induction concentrations elicited the responses required for the proper conduct of a sensitization study. The challenge responses were considered a mixture of irritation and sensitization responses, but the amount of irritation present did not confound interpretation of the data. Rechallenge with a lower concentration reduced the irritation response to a more appropriate level (as seen in the irritation control group responses) and the results of both the challenge and rechallenge revealed the test substance had moderate sensitization potential (modified from Thomas, 1970).

Executive summary:

The allergic contact sensitization potential of the test substance was evaluated in the guinea pig using the Guinea Pig Maximization Test (Magnusson and Kligman, 1969; OECD, 1992; EC, 1993). Twenty female Hartley albino guinea pigs received intradermal injection and topical exposures to the test substance. An irritation control group of 20 female guinea pigs received the induction carrier, peanut oil, through the induction phases of the study and then exposure to the test substance at challenge (ten animals) or rechallenge (ten animals). The challenge carrier, olive oil also was topically challenged or rechallenged in both the treated and irritation control groups. Evaluation of sensitization potential was based on a comparison of the treated and irritation control group dermal scores. The results of the testing facility's most recent reliability test using the positive control material 2-mercaptobenzothiazole (MIST) are reported.

Primary range-finding tests were conducted to determine appropriate concentrations of the test substance to be used in the three phases of dosing. Based on the results of these tests, concentrations of 5%, 100%, and 100% were chosen for the intradermal induction, topical induction, and topical challenge, respectively. Due to irritation observed during challenge, rechallenge was conducted at 50% test substance in olive oil to reduce the amount of background irritation.

Dermal responses were evaluated approximately 1 and 24-hours after the intradermal induction using descriptive terminology. Dermal responses were evaluated 1 and 24-hours (Days 9 and 10) after removal of the topical induction patch, and 24 and 48 hours (Days 23 and 24) after removal of the topical challenge patch, and 24 and 48 hours (Days 30 and 31) after removal of the rechallenge patch using the scoring system described in Key A. Observations were made as to the nature, onset, severity and duration of toxicological signs after dosing on Day 0 and on Days 7, 14, 21, and 28 and on Day 31. Body weights were recorded once at pretest and on Days 0, 7, 14, 21, 28 and 31. After the final observations and weighing’s, all animals were sacrificed by exsanguination following carbon dioxide asphyxiation and discarded without further examination.

All animals survived until study termination. Emaciation and/or little sign of stool were the most frequently observed clinical signs and were noted in one treated group and two irritation control group animals. All animals gained weight over their initial Day 0 values.

Intradermal induction conducted with 5% test substance did not produce eschar or necrosis. Topical induction with the neat test substance produced Grade ± to 2 responses in all animals.

Topical challenge with 100% test substance elicited responses ranging from Grade 1 to 3 in all treated animals and responses ranging from Grade ± to 2 in all irritation control animals at the 24-hour observation. Edema was noted in four treated animals at the 24-hour observation. At the 48-hour observation, the severity of the responses remained essentially the same for the treated group. The response for the irritation control group decreased slightly at the 48-hour observation and irritation was observed in eight of the ten animals. In the treated group, edema was noted in four animals and cracking was noted in three animals at the 48-hour observation.

In an attempt to reduce the irritation observed at challenge in the irritation control group, the topical rechallenge was conducted with 50% test substance in olive oil. The rechallenge elicited responses ranging from Grade ± to 2 in all treated animals at the 24-hour observation. The rechallenge with 50% test substance in olive oil elicited Grade ± and Grade1responses in two and one of the 10 irritation control animals, respectively,at the 24-hour observation: At the 48- hour observation, Grade ± responses were noted in nine treated animals, Grade 1 responses were noted in five treated animals, and Grade 2 responses in three treated animals. A Grade 1 response was noted in one of 10 irritation control animals at this interval.

Topical application of olive oil at challenge and rechallenge elicited primarily Grade ± responses but occasionally Grade 1 responses were observed. Responses generally decreased by 48 Hours at both challenge and rechallenge.

At challenge 12 out of 20 of the treated animals (60%) exhibited a reaction that could be attributed to an allergic response. At rechallenge six out of 20 of the treated animals (30%) exhibited a reaction that could be attributed to an allergic response. According to the ranking system proposed by Magnusson and Kligman (1969) the test substance would be assigned an allergenicity rating of Grade III (moderate classification)for either interval.

In conclusion, the induction concentrations elicited the responses required for the proper conduct of a sensitization study. The challenge responses were considered a mixture of irritation and sensitization responses, but the amount of irritation present did not confound interpretation of the data. Rechallenge with a lower concentration reduced the irritation response to a more appropriate level (as seen in the irritation control group responses) and the results of both the challenge and rechallenge revealed the test substance had moderate sensitization potential (modified from Thomas, 1970).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance was tested in a Guinea-Pig Maximisation Test, which employs intradermal injection and use of adjuvant. Upon challenge application, six of the 20 animals treated with the test substance showed slight and patchy erythema; the control animals showed no response to challenge. Therefore the substance was concluded to be a weak sensitiser in this test. In accordance with CLP, when an adjuvant type guinea pig test is used, a response of at least 30% of the animals (equivalent to six out of 20 animals) is considered as positive. Therefore, the substance is classified as a skin sensitiser (Category 1B) under CLP.

There are no human or suitable animal data regarding the respiratory sensitisation potential of the substance. As the substance is only a borderline skin sensitiser according to CLP criteria, and as the potential for inhalation exposure to the substance will be negligible, no classification is proposed for respiratory sensitisation.