[No public or meaningful name is available]

Endpoint summary

Description of key information

non-sensitising, Guinea Pig, OECD TG 406, 2000

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:

17-04-2000 to 17-07-2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. All relevant validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

Method employed in this study for the detection of delayed contact hypersensitivity was the guinea-pig maximization test described by B. Magnusson and A.M. Kligman - "The identification of contact allergens by animal assay, the guinea pig maximisation method"; Invest. Dermatol. 1969. 52, 268-276

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147 (2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: February 2000 ; signature: April 2000

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Scientific justification and information as to the availability of the study is attached by the applicant in 'attached background material'.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier
- Age at study initiation: Young adult (approximately 8 - 12 weeks old).
- Weight at study initiation: 351 – 447 g
- Housing: Individually or in pairs in solid-floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): Complete maintenance diet for guinea pigs (details in the full study report).
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions; identical to the test

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C
- Humidity (%): 30 – 70%
- Air changes (per hr): at least 15 per hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark

IN-LIFE DATES: From: To: 17-04-2000 to 03-06-2000

Route:

intradermal

Vehicle:

arachis oil

Concentration / amount:

- Intradermal: 5% test item in Arachis Oil BP

Day(s)/duration:

Day 0

Adequacy of induction:

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

Route:

epicutaneous, occlusive

Concentration / amount:

- Topical: 100% test material (undiluted)

Day(s)/duration:

Day 7

Adequacy of induction:

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

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

arachis oil

Remarks:

Arachis Oil BP for the intradermal injections and in Arachis Oil BP for the topical applications (as applicable)

Concentration / amount:

- Challenge: 75% in Arachis Oil BP and 50% in Arachis Oil BP

Day(s)/duration:

Day 21

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Test group: 20 ; Control group: 10

Details on study design:

RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test item concentrations to be used in the main study. Intradermal induction: two concentrations were investigated: 1% and 5% v/v in Arachis Oil BP. Two males were used each receiving four 0.1 mL injections of only one concentration of test item. The degree of erythema at the injection sites was assessed approximately 24, 48 and 72 hours and 7 days after injection. Topical Induction: Two males (intradermally injected with Freund's Complete
Adjuvant nine days earlier) were treated with the undiluted test Item and three preparations of the test material (75%, 50% and 25% v/v in arachis oil BP). Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. Topical Induction: The undiluted test material and three preparations of the test item (75%, 50% and 25% v/v in arachis oil BP) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after
dressing removal. The highest non-irritant concentration of the test item and one lower concentration were selected for the topical challenge stage of the main study.

Final concentrations for definitive testing based on preliminary irritation study:
- Intradermal: 5% test item in Arachis Oil BP
- Topical: 100% test item (undiluted)
- Challenge: 75% and 50% in Arachis Oil BP
% are percent v/v of test item in vehicle. Vehicle: Arachis Oil BP (intradermal induction and topical, as applicable), was chosen on the basis of the most suitable formulation at the required concentrations and maximising the solubility of the test item.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal induction; 1 epidermal induction (topical booster)
- Exposure period: Day 0 intradermal induction and Day 7 topical induction (topical booster)
- Test groups: duplicate injections as follows: 2 ID: Freund's Complete Adjuvant diluted at 50% in water; 2 ID: test item at 5% in arachis oil ; 2 ID: a test item at 5% in arachis oil in equal volumes v/v of FCA at 50% and water at 50%
- Control group: Vehicle and FCA only.
- Site: intradermal induction – three pairs of injections in clipped scapular region;
- Frequency of applications:
- Duration: 0-7 days. On day 7, shorn topical application of 100% test item. On day 7 : 48 hours for epidermal induction. The dressing was removed after 48 hours exposure
- Concentrations: Intradermal induction: A) A 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection; B) The test item at 5% concentration ; C) A 1:1 w/w mixture of the test item, at 5% and Freunds' Complete Adjuvant plus water (50:5)0. Topical induction: 100% test item concentration using occlusive dressing.
The control group were treated as described for the experimental group except that, instead of the test item, the vehicle was administered.

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 24 hours (topical challenge)
- Exposure period: Day 21 the dressing was removed after 24 hours exposure. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.
- Test groups: 1; test item 75% in Arachis Oil BP vehicle (and 50%) within the same test group
- Control group: 1; vehicle only
- Site: One flank (clipped)
- Concentrations: 75% and 50% in Arachis Oil BP using occlusive dressing.
- Evaluation (hr after challenge): 24 and 48 hours after dressing removal (at Day 23 and 24).
The control group were treated as described for the experimental group except that, instead of the test substance, the vehicle was administered.

OTHER: Mortality, toxicity and body weights along with irritation were examined as part of the study.

Challenge controls:

(Naive) negative control groups consisting of 10 males were exposed to the vehicle in the induction and challenge, consistent the main study with the difference that instead of test item only the vehicle was administered during induction.

Positive control substance(s):

yes

Remarks:

2-Mercaptobenzothiazole (5 and 10% ; no concurrent)

Positive control results:

A reliability check was performed (six tests are listed with at least 6, 12 and 18 months conduct prior to the study and presented in the full study report) to check the sensitivity of the test system and the reliability of the experimental techniques used. The study used the same conditions as the main study using 2-Mercaptobenzothiazole (at 5 and 10 concentrations %v/v) as positive control.
The skin reactions observed in seven experimental animals in response to the > 20 % test substance concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 70 - 100% to the 10 %w/w concentration.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

75%

No. with + reactions:

0

Total no. in group:

19

Clinical observations:

Total no. in groups: 20.0 ; Number 5 terminated due to ill health on day 9 ; Clinical observations: None reported; maximum score = 0 (no visible change).

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

75%

No. with + reactions:

0

Total no. in group:

19

Clinical observations:

Total no. in groups: 20.0 ; Number 5 terminated due to ill health on day 9 ; Clinical observations: None reported; maximum score = 0 (no visible change).

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

9

Clinical observations:

Total no. in groups: 100.0 ; Number 22 found dead on day 9 ; Clinical observations: None reported; maximum score = 0 (no visible change).

Remarks on result:

no indication of skin sensitisation

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

9

Clinical observations:

Total no. in groups: 100.0 ; Number 22 found dead on day 9 ; Clinical observations: None reported; maximum score = 0 (no visible change).

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

No. with + reactions:

7

Total no. in group:

15

Clinical observations:

Non-concurrent Positive Control Groups: Total no. in groups: 15 ; minimum score = 70% and maximum score = 100% (details in the full study report)

Remarks on result:

positive indication of skin sensitisation

Table 1: Preliminary irritation study

Skin reactions after intradermal injection

Identification		Concentration (% v/v)	Erythema grading	Systemic Toxicity
A (male)	24 hours	1	2	None
	48 hours		2	None
	72 hours		2	None
	7 days		1-2	None
B (male)	24 hours	5	2	None
	48 hours		2	None
	72 hours		2	None
	7 days		1-2	None

The concentration selected for the intradermal injection stage was 5% v/v in Arachis Oil BP

 

Table 2: Preliminary irritation study

Skin reactions after topical application

Identification	Concentration (% v/v)	Skin Reactions (hours after removal of patches)
		1			24			48
		Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
C (male)	100	2	0	-	1	0	-	0	0	-
	75	2	0	-	1	0	-	0	0	-
	50	2	0	-	0	0	-	0	0	-
	25	1	0	-	0	0	-	0	0	-
D (male)	100	2	0	-	1	0	-	0	0	-
	75	2	0	-	0	0	-	0	0	-
	50	1	0	-	0	0	-	0	0	-
	25	1	0	-	0	0	-	0	0	-

Where:

Er = erythema

Oe = oedema

- = no other reactions noted

The undiluted (100%) test item was selected for topical induction

 

Table 3: Preliminary irritation study

Topical application for challenge application

Identification	Concentration (% v/v)	Skin Reactions (hours after removal of patches)
		1			24			48
		Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
E (male)	100	2	0	-	1	0	-	0	0	-
	75	1	0	-	0	0	-	0	0	-
	50	1	0	-	0	0	-	0	0	-
	25	1	0	-	0	0	-	0	0	-
F (male)	100	1	0	-	1	0	-	0	0	-
	75	1	0	-	0	0	-	0	0	-
	50	1	0	-	0	0	-	0	0	-
	25	1	0	-	0	0	-	0	0	-

The concentration of test item for topical challenge was 75% and 50% v/v in Arachis Oil BP

 

Table 4: Challenge applications

Identification	Skin Reactions (hours after removal of patches)
	24						48
	50%			75%			50%			75%
	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
1	0	0	-	0	0	-	0	0	-	0	0	-
2	0	0	-	0	0	-	0	0	-	0	0	-
3	0	0	-	0	0	-	0	0	-	0	0	-
4	0	0	-	0	0	-	0	0	-	0	0	-
5	*	*	*	*	*	*	*	*	*	*	*	*
6	0	0	-	0	0	-	0	0	-	0	0	-
7	0	0	-	0	0	-	0	0	-	0	0	-
8	0	0	-	0	0	-	0	0	-	0	0	-
9	0	0	-	0	0	-	0	0	-	0	0	-
10	0	0	-	0	0	-	0	0	-	0	0	-
11	0	0	-	0	0	-	0	0	-	0	0	-
12	0	0	-	0	0	-	0	0	-	0	0	-
13	0	0	-	0	0	-	0	0	-	0	0	-
14	0	0	-	0	0	-	0	0	-	0	0	-
15	0	0	-	0	0	-	0	0	-	0	0	-
16	0	0	-	0	0	-	0	0	-	0	0	-
17	0	0	-	0	0	-	0	0	-	0	0	-
18	0	0	-	0	0	-	0	0	-	0	0	-
19	0	0	-	0	0	-	0	0	-	0	0	-
20	0	0	-	0	0	-	0	0	-	0	0	-
21 (control group)	0	0	-	0	0	-	0	0	-	0	0	-
22	#	#	#	#	#	#	#	#	#	#	#	#
23	0	0	-	0	0	-	0	0	-	0	0	-
24	0	0	-	0	0	-	0	0	-	0	0	-
25	0	0	-	0	0	-	0	0	-	0	0	-
26	0	0	-	0	0	-	0	0	-	0	0	-
27	0	0	-	0	0	-	0	0	-	0	0	-
28	0	0	-	0	0	-	0	0	-	0	0	-
29	0	0	-	0	0	-	0	0	-	0	0	-
30	0	0	-	0	0	-	0	0	-	0	0	-

Where:

* = no data ; terminated on day 9 due to ill health

# = no data ; mortality on day 9

Interpretation of results:

GHS criteria not met

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this study, the test material is not considered to be a contact sensitizer.

Executive summary:

The study was performed according to a method equivalent to guideline OECD TG 406 and EU Method B.6 and consistent with Magnusson-Kligman Guinea Pig Maximisation test under GLP to assess the skin sensitisation potential of the test item. Concentrations selected for the main study were based on the results of a preliminary study. In the main study, after induction (intradermic injection at 5% v/v in Arachis Oil BP and topical application at 100%) and a ten day rest phase of twenty experimental animals were challenged with 75% and 50% v/v in Arachis Oil BP along with parallel negative control group of ten animals: challenged at 75% and 50% v/v in Arachis Oil BP. The treated group (challenge treatment doses of 75%v/v and/or 50%v/v in Arachis Oil BP), gave no skin reactions at 24 and 48-hour observation. No skin reactions were noted in the control groups. There was no test item treatment related mortality. All surviving animals gained bodyweight at the end of the study. Under the conditions of this study, the test item is not considered to be a contact skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Key Study : OECD TG 406, 2000 : The study was performed according to a method equivalent to guideline OECD TG 406 and EU Method B.6 and consistent with Magnusson-Kligman Guinea Pig Maximisation test under GLP to assess the skin sensitisation potential of the test item. Concentrations selected for the main study were based on the results of a preliminary study. In the main study, after induction (intradermic injection at 5% v/v in Arachis Oil BP and topical application at 100%) and a ten day rest phase of twenty experimental animals were challenged with 75% and 50% v/v in Arachis Oil BP along with parallel negative control group of ten animals: challenged at 75% and 50% v/v in Arachis Oil BP. The treated group (challenge treatment doses of 75%v/v and/or 50%v/v in Arachis Oil BP), gave no skin reactions at 24 and 48-hour observation. No skin reactions were noted in the control groups. There was no test item treatment related mortality. All surviving animals gained bodyweight at the end of the study. Under the conditions of this study, the test item is not considered to be a contact skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In the GPMT test the severity of effects were non-existent (score = 0) at 24 and 48 hours in nineteen organisms when topically challenged at up to the maximum non-irritating concentration 75%v/v and 50%v/v (Arachis Oil BP) following intradermal induction at 5%v/v (Arachis Oil BP) with topical booster at 100%. The test was consistent with established adjuvant-test methodology. Under EU criteria there was no or only very limited effects observed in the in vivo GPMT study on exposure to the substance at > 1% v/v intradermal induction dose, which hare inadequate for classification. The substance cannot be considered a skin sensitiser as there is no evidence of significant effects in available animal studies and no history of the test item yielding positive responses in humans.

 

The overall weight of evidence is indicative of the substance not meeting the relevant classification criteria.

 

References:

1. ECHA Guidance on Application on the CLP Criteria, section 3.4.2.2 (v5.0, July 2017)