Ferbam

Endpoint summary

Description of key information

Skin sensitisation: Split Adjuvant Test, 20 female guinea pigs at 25%, 10% and 5% w/w in 1% aqueous methyl cellulose, results: one animal showed positive skin reactions (sensitisation rate of 5%). It can be expected that the weak sensitisation resulted from the Thiram impurity of the test item.

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:

1987-11-16 - 1987-12-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 81-6 (Skin Sensitisation)

Version / remarks:

revised Nov. 1984

Deviations:

no

GLP compliance:

yes

Remarks:

US FDA (21 CFR 58) and US EPA (40 CFR 160 and 40 CFR 792)

Type of study:

split adjuvant test

Justification for non-LLNA method:

The guinea pig is the classical animal for determining the potential of substance to induce delayed contact hypersensitivity.
The dermal route of administration was selected because the test substance may accidentally come into contact with the skin during manufacture, handling or use.
Freunds Complete Adjuvant (FCA) is intracutaneoulsy injected to attract circulating immunocompetent cells to the epicutaneously exposed area.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Thirty-five young adult female guinea pigs of the Dunkin-Hartley strain, approximately 2 months old at the start of the experiment, SPF-quality, were obtained from Charles River Wiga, FRG. Date of arrival at the animal house: November 3, 1987. The animals were nulliparous and non-pregnant; they were identified by means of tattoo behind the ears. A few days before the start of the experiment, the body weights ranged from 357 to 443 g (see Tables 1 and 2). The animals were housed in metal cages with wire-mesh floors (ITL, Bergen, The Netherlands: 2 animals per cage). They were fed standard guinea pig diet, including ascorbic acid (1600 mg/kg) obtained from Hope Farms, Woerden (LC 23-B, pellet diameter 4 mm) and had free access to tap-water. In addition, once a week hay was provided.

ENVIRONMENTAL CONDITIONS
The animal room temperature was between 19 and 23 °C and the relative humidity between 55 and 95 per cent. The artificial light sequence was 12 hours light, 12 hours dark. A combined quarantine/acclimation period of 13 days was allowed (7 days for the animals of the primary irritation experiment).

Route:

epicutaneous, occlusive

Vehicle:

other: 1% aqueous methyl cellulose

Concentration / amount:

25% in 0.2 mL

Day(s)/duration:

20 days

Adequacy of induction:

other: highest concentration used causing no substantial skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

other: 1% aqueous methyl cellulose

Concentration / amount:

25%, 10%, 5%, 0% in 0.5 mL

Day(s)/duration:

1 - 3 days

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Primary irritation experiment: 4 (+1evaluation of toxicity)
Experimental group: 20 females
Control group: 10 females

Details on study design:

RANGE FINDING TESTS:

Primary irritation experiment
A primary irritation test was carried out using five guinea pigs to determine the concentrations to be applied in the challenge phase (these should not produce any substantial irritation). Four guinea pigs were shaved and their flank was exposed for 24 hours to 50%, 25%, 10% and 5% (W/W) of the test substance in 1% aqueous methyl cellulose, occlusively administered by means of four Square chambers (v.d. Bend, Brielle, The Netherlands). None of the animals reacted to these test substance concentrations.
One additional animal received 0.5 ml of the test substance, 50% in 1% methyl cellulose, on a patch of Metalline (Lohmann. W.-Germany) for evaluation of the toxicity of the compound. In this case, also no irritation was observed. No systemic toxic effects resulted from these treatments in any of the animals.

MAIN STUDY
A. INDUCTION EXPOSURE
The experimental animals were clipped and shaved in an area of skin on the back just behind the right shoulder girdle. Each animal was wrapped in a "window dressing" which consisted of Coban elastic bandage (Smith and Nephew, England). A 2 x 2 cm opening was cut in the dressing over the exposure site. Dry ice was applied to the test site for approximately 5 sec with firm pressure. Then 0.2 ml of test substance, 25% (W/W) in 1% methyl cellulose was applied, and this was covered with a Metalline-patch (Lohmann). Then the patch was covered with water-impervious tape (Sleek, Smith and Nephew, England).
On day 2, the dressing that covered the window was removed, 0.2 ml of the test material in 1% methyl cellulose was applied, and the window was reclosed with the same dressing.
On day 4, the window was again opened and 0.1 ml of Freund's complete adjuvant (Difco, Detroit, USA) was injected intradermally on both sides of the exposure site. This was followed by a further application of 0.2 ml of the test substance in 1% aqueous methyl cellulose, and the window was closed. On day 7, the test material was again applied. On day 9, all wrappings were removed. On day 4, the negative control animals received i.d. injections with Freund's complete adjuvant to exclude interpretation difficulties in the challenge phase caused by aspecific lowering of the skin irritation threshold by this substance.

B. CHALLENGE EXPOSURE
On day 21, the flank was carefully shaved and the protruding long hairs from the edges were cut away with a pair of scissors. The test substance, suspended in 1% methyl cellulose, was applied in a volume of 0.05 ml on Square chambers (v.d. Bend, Brielle, The Netherlands), mounted on Micropore-tape. Each animal received 4 different doses of test substance; all animals were treated similarly. The concentrations tested were: 25%, 10%, 5% and 0% (W/W) of the test substance in 1% aqueous methyl cellulose. The elastic bandage (Coban) was kept in place for 24 hours. Twenty-four hours after removal of the dressings the first reading was made after closely shaving the skin in the morning.
Further readings were made 48 and 72 hours after removal of the bandage. If necessary for evaluation of the skin reactions, shaving was repeated.

Positive control substance(s):

yes

Remarks:

formaldehyde solution 37%

Positive control results:

A sensitisation rate of 50 per cent was obtained to the 0.5% concentration.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25 %, 10 %, 5 %

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

First challenge: Skin reactions of grade 1 were observed in one experimental animal. No skin reactions were evident in the control animals.

Remarks on result:

other: weak sensitising properties

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

25 %, 10 %, 5 %

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

Second challenge: Skin reactions of grade 1 were observed in one experimental animal. No skin reactions were evident in the control animals.

Remarks on result:

other: weak sensitising properties

Key result

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

25 %, 10 %, 5 %

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

Third challenge: Skin reactions of grade 1 were observed in one experimental animal. No skin reactions were evident in the control animals.

Remarks on result:

other: weak sensitising properties

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0 %

No. with + reactions:

1

Total no. in group:

10

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:

10

Remarks on result:

no indication of skin sensitisation

Reading:

other: 3rd reading

Hours after challenge:

72

Group:

negative control

Dose level:

0 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Group:

positive control

Remarks on result:

not measured/tested

The test substance, suspended in 1% aqueous methyl cellulose at several concentrations (25%, 10% and 5%) and administered epicutaneously to guinea pigs in the challenge phase, resulted in positive skin reactions (grade 2) in one experimental animal (no. 49). No positive reactions were observed in animals of the control group.

Table 1: Primary Irritation Experiment: Erythema* from exposure of the guinea pig skin to the test material for 24 hours
Animal #	Day -6 BW** (gram)	24 Hours readings***				48 Hours readings				Day 2 BW (gram)
		50****	25	10	5	50****	25	10	5
66	381	0	0	0	0	0	0	0	0	423
67	368	0	0	0	0	0	0	0	0	407
68	359	0	0	0	0	0	0	0	0	384
69	384	0	0	0	0	0	0	0	0	400
* Grading scheme: see below ** body weight *** Time after removal of dressings **** Concentration in 1 % aqueous methyl cellulose (% w/w)
Erythema				Grade
no erythema				0
very slight erythema				1
well-defined erythema				2
moderate to severe erythema				3
severe erythema (beet redness)				4

Table 2: Individual body weights and skin readings of guinea pigs challenged with the test material
Group	Animal #	Day - 3 BW* (gram)	Day 23 Readings				Day 24 Readings				Day 25 Readings				Day 25 BW* (gram)	Comments
			25**	10	5	0	25**	10	5	0	25**	10	5	0
Expr.	36	383	0	0	0	0	0	0	0	0	0	0	0	0	532	not sensitised
	37	381	0	0	0	0	0	0	0	0	0	0	0	0	516	not sensitised
	38	429	0	0	0	0	0	0	0	0	0	0	0	0	587	not sensitised
	39	378	0	0	0	0	0	0	0	0	0	0	0	0	503	not sensitised
	40	413	0	0	0	0	0	0	0	0	0	0	0	0	563	not sensitised
	41	430	0	0	0	0	0	0	0	0	0	0	0	0	594	not sensitised
	42	406	0	0	0	0	0	0	0	0	0	0	0	0	598	not sensitised
	43	423	0	0	0	0	0	0	0	0	0	0	0	0	578	not sensitised
	44	357	0	0	0	0	0	0	0	0	0	0	0	0	747	not sensitised
	45	358	0	0	0	0	0	0	0	0	0	0	0	0	500	not sensitised
	46	373	0	0	0	0	0	0	0	0	0***	0	0	0	494	not sensitised
	47	379	0	0	0	0	0	0	0	0	0	0	0	0	525	not sensitised
	48	370	0	0	0	0	0	0	0	0	0	0	0	0	477	not sensitised
	49	366	0	1	1	0	2	2	2	0	2***	2	2	0	516	sensitised
	50	399	0	0	0	0	0	0	0	0	0	0	0	0	554	not sensitised
	51	372	0	0	0	0	0	0	0	0	0	0	0	0	510	not sensitised
	52	415	0	0	0	0	0	0	0	0	0	0	0	0	571	not sensitised
	53	430	0	0	0	0	0	0	0	0	0	0	0	0	568	not sensitised
	54	367	0	0	1	0	0	0	0	0	0	0	0	0	504	not sensitised
	55	408	0	0	1	0	0	0	0	0	0	0	0	0	556	not sensitised
Contr.	56	404	0	0	0	0	0	0	0	0	0	0	0	0	580
	57	383	1	0	0	0	0	0	0	0	0	0	0	0	566
	58	357	0	0	0	0	0	0	0	0	0	0	0	0	448
	59	367	0	0	1	0	0	0	0	0	0	0	0	0	494
	60	406	0	0	0	0	0	0	0	0	0	0	0	0	585
	61	398	0	0	1	0	0	0	0	0	0	0	0	0	482
	62	387	0	0	0	0	0	0	0	0	0	0	0	0	529
	63	387	0	0	0	0	0	0	0	0	0	0	0	0	552
	64	443	0	0	0	0	0	0	0	0	0	0	0	0	478
	65	363	0	0	0	0	0	0	0	0	0	0	0	0	575
* body weight ** Concentration in 1 % aqueous methyl cellulose (% w/w) *** this site had ascaly appearance

Interpretation of results:

other: Category 1 (skin sensitising) based on EU GHS criteria

Conclusions:

A sensitisation rate of 5 % was obtained. Based on this sensitisation rate the test substance should be considered to have weak sensitising properties.

Executive summary:

The test material was tested in the Split Adjuvant Test according to EPA OPP 81-6 (Skin Sensitisation) and in compliance with GLP to determine its sensitizing potential to induce delayed contact hypersensitivity in the guinea pig.

In a primary irritation experiment with five adult female guinea pigs the concentrations to be applied in the challenge phase was determined (these should not produce any substantial irritation) at concentrations of 5%, 10%, 25% and 50% w/w.

In the main experiment 20 adult female guinea pigs (Dunkin-Hartley strain) were induced by epicutaneous applications of the test substance, 25% (w/w) in 1% aqueous methyl cellulose. After being challenged with a series of test substance concentrations (25 %, 10% and 5%, w/w) in 1% aqueous methyl cellulose, one of the experimental animals showed a weak positive skin reaction to all three concentrations tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation

The test material was tested in the Split Adjuvant Test (Weterings, 1987c) according to EPA OPP 81-6 (Skin Sensitisation) and in compliance with GLP to determine its sensitising potential to induce delayed contact hypersensitivity in the guinea pig.

In a primary irritation experiment with five adult female guinea pigs the concentrations to be applied in the challenge phase was determined (these should not produce any substantial irritation) at concentrations of 5%, 10%, 25% and 50% w/w. in the main experiment 20 adult female guinea pigs (Dunkin-Hartley strain) were induced by epicutaneous applications of the test substance, 25% (w/w) in 1% aqueous methyl cellulose. After being challenged with a series of test substance concentrations (25 %, 10% and 5%, w/w) in 1% aqueous methyl cellulose, one of the experimental animals showed a positive skin reaction to all three concentrations tested. Thus, a sensitisation rate of 5 % was obtained which is most likely caused by the Thiram impurity which is a known skin sensitiser (Cat 1 harmonised classification - Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation)).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test material showed a sensitisation rate of 5% in an GLP compliant Split Adjuvant Test.

Therefore, the test item is regarded as a weak sensitser and meets the criteria for classification and labelling for skin sensitisation (Cat.1) in accordance with Regulation (EC) No 1272/2008 (CLP Regulation).