O,O-tert-butyl isopropyl monoperoxycarbonate

Endpoint summary

Description of key information

The substance is a moderate skin sensitizer, Cat 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:

Start: 19 October 1998 End: 27 November 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was conducted in 1998
The purpose of this study was to evaluate whether the test substance induces contact hypersensitivity in guinea pigs after intradermal and epidermal exposure of the animals under the conditions described in this report.
This study should provide a rational basis for risk assessment in man.
The Maximisation test is selected because it is regarded as the most sensitive and the preferred method with regard to testing for sensitisation potential.

Specific details on test material used for the study:

Batch: 0419804130350
Test substance storage: In refrigerator in the dark. Do not heat test substance.
Stability under storage conditions: Not indicated
Expiry date: 01 October 1999 (allocated by NOTOX, 1 year after receipt of the test substance)
Density: 900-910 kg/m^3 (20°C)
Stability in vehicle Corn oil: at least 96 h
Vehicle: Corn oil
Rationale: The vehicle was selected based on a pretest performed at NOTOX.
Preparation: When required, the test substance formulations (w/w) were prepared prior to each treatment. No adjustment was made for specific gravity of vehicle. Homogeneity was obtained to visually acceptable levels.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Species: Dunkin Hartley strain, albino guinea pig (SPF-quality). Recognised by international guidelines as the recommended test system (e.g. OECD, EC).
Source: Charles River, Germany.
Number of animals: Experimental group: 10 females.
Control group: 5 females. (females were nulliparous and non-pregnant).
Age and body weight: Young adult animals (approx. 5 weeks old) were selected. Individual body weights did not exceed 500 grams.
Identification: Ear tattoo.
Reliability check: The results of a reliability test performed not more than 6 months previously are summarised in the Appendix. Similar procedures were used in the reliability test and in this study.

ANIMAL HUSBANDRY
Conditions:
Air -conditioned room with approximately 15 air changes per hour and the environment controlled with optimal conditions considered as being a temperature of 21°C and a relative humidity of 50%. Fluctuations from these optimal conditions were noted, but were considered not to have affected study integrity. Lighting was 12 hours artificial fluorescent light and 12 hours dark per day.

Accommodation
Group housing of 5 animals per labelled metal cage with wire-mesh floors and equipped with an automatic drinking system (ITL, Bergen, The Netherlands). The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions.

Diet
Free access to standard guinea pig diet, including ascorbic acid (1600 mg/kg); LC 23-B, pellet diameter 4mm (Hope farms, Woerden, The Netherlands). Certificates of analysis were examined and retained in the NOTOX archives.
In addition, hay (B.M.I., Helmond, The Netherlands) was provided once a week.

Water
Free access to tap-water, diluted with decalcified water. Certificates of quarterly analysis for tap-water were examined and retained in the NOTOX archives.

Route:

intradermal

Vehicle:

corn oil

Concentration / amount:

10% concentration

Day(s)/duration:

8 days

Adequacy of induction:

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

Route:

intradermal

Vehicle:

water

Remarks:

Mixture with Freunds' Complete Adjuvant (Difeo, Detroit, U.S. A.)

Concentration / amount:

1:1 (w/w) mixture

Day(s)/duration:

8 days

Adequacy of induction:

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

Route:

intradermal

Vehicle:

other: Freunds' Complete Adjuvant

Concentration / amount:

1:1 (w/w) mixture with 20% concentration of test material

Day(s)/duration:

8 days

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:

0.5ml undiluted test substance

Day(s)/duration:

48 hrs

Adequacy of induction:

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

No.:

#1

Route:

other: epicutaneuous - Patch Test Plasters

Vehicle:

corn oil

Concentration / amount:

20% test substance concentration and the vehicle (0.15 ml each)

Day(s)/duration:

Day 22. The dressing was removed after 24 hours exposure.

Adequacy of challenge:

highest non-irritant concentration

No.:

#2

Route:

other: epicutaneous - Patch Test Plasters

Vehicle:

corn oil

Concentration / amount:

10% and 5% test substance concentration

Day(s)/duration:

Day 29

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

10 animals per dose

Details on study design:

PRELIMINARY IRRITATION STUDY
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the Main Study. The selection of concentrations was based on the following criteria:
- The concentrations are well-tolerated by the animals.
- For the induction exposures: the highest possible concentration that produced mild to moderate irritation (grades 2 - 3).
- For challenge exposure: the maximum non-irritant concentration.

Series of test substance concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1% and, if needed, further lower concentrations using the same steps.
The test system, procedures and techniques were identical to those used during the main study, unless otherwise specified.
The animals were between 4 and 9 weeks old, and as a consequence the body weights may exceed 500 grams. Body weights were determined prior to treatment.

Intradermal injections:
A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 ml/site) in the clipped scapular region. If possible, the injection sites were assessed for irritation 24 and 48 hours after treatment.

Epidermal application:
A series of four test substance concentrations was used, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 ml each) per animal to the clipped flank, using Metalline patches' (2x3 cm) mounted on Medical tape, which were held in place with Micropore tape· and subsequently Coban elastic bandage. The animals receiving intradermal injections were treated with the lowest concentrations and two further animals with the highest concentrations.
After 24 hours, the dressing was removed and the skin cleaned of residual test substance.
If possible, the treated skin areas were assessed for irritation 24 and 48 hours after exposure.
' Supplier: Lohmann GmbH, Neuwied, Germany
' Supplier: 3M, St. Paul, U.S.A.

INDUCTION - Experimental animals
Day 1 The scapular region was clipped and three pairs of intradermal injections (0. 1 ml/site) were made in this area as follows:
A) A 1:1 w/w mixture of Freunds' Complete Adjuvant (Difeo, Detroit, U.S. A.) with water for injection (Fresenius AG, Bad Homburg, Germany).
B) The test substance at a 10% concentration.
C) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial A) to caudal C).

Day 3 The dermal reactions caused by the intradermal injections were assessed for irritation.
Day 8 The scapular area between the injection sites was clipped and subsequently treated with 0.5 ml of the undiluted test substance using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.
The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance and the dermal reactions caused by the epidermal exposure were assessed for irritation.
INDUCTION · Control animals
The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.

CHALLENGE · Control and experimental animals
Day 22 One flank of the animals was clipped and treated by epidermal application of a 20% test substance concentration and the vehicle (0.15 ml each), using Patch Test Plasters (Leukoteste, Beiersdorf Medical, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage.
The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.
Day 29 A re-challenge was conducted approximately one week after the first challenge, to clarify the results in the first challenge. The contralateral flank of the animals was similarly treated, except that the animals were treated epidermally with a 10% and a 5% test substance concentration.

OBSERVATIONS
Mortality/Viability: Twice daily
Toxicity: At least once daily.
Body weights: Prior to start and at termination of the study.
Necropsy: The moribund animal was subjected to necropsy for gross macroscopic examination.
Irritation: Skin reactions were graded according to the following numerical scoring systems. Furthermore, a description of all other (local) effects was recorded. Whenever necessary, the treated skin-areas were clipped at least 3 hours before the next skin reading to facilitate scoring.

Grading Irritation Reactions':
Erythema and eschar formation:
No erythema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Slight erythema (barely perceptible) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Well defined erythema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Moderate· erythema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) . . . . . . . . . . . 4

Oedema formation:
No oedema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Slight oedema (barely perceptible) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Well-defined oedema (edges of area well-defined by definite raising) . . . . . . . . . . . . . . . . . . . 2
Moderate oedema (raised approximately 1 millimeter) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Severe oedema (raised more than 1 millimeter and extending beyond the area of exposure) . . . . . . . 4
• Intradermal reactions were assessed for erythema only or, if necrosis is present, the diameter of necrosis.

Grading Challenge Reactions:
No visible change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0
Discrete or patchy erythema . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Moderate and confluent erythema . . . . . . . . . . . . . . . . . . . . . . . . . 2
Moderate erythema and swelling . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Intense erythema and swelling . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

After the end of the study all animals were killed by asphyxiation using anoxygen/carbon dioxide procedure.

Challenge controls:

The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.

Positive control substance(s):

no

Positive control results:

no positive control

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

20% test substance concentration

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

Animal 28 was removed from the study on day 10 following the observation of emaciation, dark eyes and labored respiration.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0.15 mL of vehicle (corn oil)

No. with + reactions:

2

Total no. in group:

5

Clinical observations:

Skin reactions of grade 1 were observed in control animals.

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

10% test substance concentration

No. with + reactions:

7

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

5% test substance concentration

No. with + reactions:

5

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

0.15 mL vehicle only (corn oil)

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Group:

positive control

Remarks on result:

other: no positive control group

PRELIMINARY IRRITATION STUDY

The results of the intradermal injections and epidermal exposures for the selection of suitable test substance concentrations for the main study are described in Table 1.

Table 1: SKIN REACTIONS AFTER INTRADERMAL INJECTION

Animal Number	Conc. %	Time after injection
		24 hours		48 hours
		Erythema (grade)	Necrosis (mm)	Erythema (grade)	Necrosis (mm)
21	100		Above 10
	50		Above 10
22	20		4		5
	10	3		2

SKIN REACTIONS AFTER EPIDERMAL EXPOSURE

Animal Number	Body Weight (gram)	Conc. %	Time after injection
			24 hours		48 hours
			Erythema (grade)	Necrosis (mm)	Erythema (grade)	Necrosis (mm)
19	462	100	1	0	0	0
		50	2	0	0	0
20	494	100	3	0	2	0
		50	2	0	1	0
21*	435	20
		10
22	471	20	0	0	0	0
		10	1	0	0	0

*Based on the severity of the skin reactions, animal 21 was sacrificed for humane reasons, 24 hours after injection.

Note: It was noted that the identification of the animals used in the preliminary irritation test was identical to some animals in the main study. The raw data of the study specifies that they were of separate deliveries from the supplier.

Based on the results the test substance concentrations selected for the Main Study were a 10% concentration.for the intraderma1 induction and the undiluted test substance for the epidermal induction exposure.

A 20% test substance concentration was selected for the challenge phase.

MAIN STUDY

Induction phase

The skin effects caused by the intradermal injections and epidermal exposure during the induction phase are given in Table 2.

INDUCTION READINGS

	Animal Number	Intradermal Injection (Day 3)			Epidermal Exposure (Day 10)
		A	B	C	D
					Erythema	Oedema
Control	16	E2	NA	E2	0	0
	17	E2	E1	E2	0	0
	18	E2	E1	E2	0	0
	19	E2	E1	E2	0	0
	20	E2	NA	E2	0	0
Experimental	21	E3	E2	E3	2	0
	22	E3	N1	E2	2	0
	23	E3	N2	E3	4	0
	24	E3	N1	E3	2	0
	25	E3	N1	E2	2	0
	26	E4	N1	E2	3	0
	27	E3	N2	E2	2	0
	28	E3	N2	E3	2	0
	29	E4	N1	E3	3	0
	30	E3	N2	E3	3	0

A. 1: 1 Mixture of FCA and water for injection.

B. A 10% test substance concentration (Experimental); vehicle (Control).

C. 1:1 Mixture of FCA and a 20% test substance concentration (Experimental) or vehicle (Control).

D. Undiluted test substance (Experimental); vehicle (Control).

Skin effects intradermal injections:

NA No abnormalities

E (.) Erythema (grade)

N (.) Signs of necrosis (mm in diameter)

Challenge phase

First Challenge.

Skin reactions of grade 1 were observed in experimental and control animals in response to the 20% test substance concentration (see Table 3). Scaliness was seen in the treated skin site of one experimental animal.

Table 3: FIRST CHALLENGE READINGS

	Animal No.	Day 24 Readings		Day 25 Readings		Comments
		20%^#	Vehicle	20%	Vehicle
Control	16	1	0	1	0
	17	1	0	1	0
	18	0	0	0	0
	19	0	0	0	0
	20	0	0	0	0
Experimental	21	0	0	0	0
	22	0	0	1	0
	23	1	0	0	0
	24	0	0	0	0
	25	1	0	0	0
	26	1	0	0	0
	27	0	0	0	0
	28*
	29	1	0	1p	0
	30	0	0	0	0

#. Test substance concentration.

p. Scaliness.

* . Animal 28 was removed from the study on day 10 following the observation of emaciation, dark eyes and labored respiration.

Second challenge.

Since comparable skin reactions were observed in both experimental and control animas, a second cha11enge was performed one week 1ater. The animals were then treated with a 10% and a 5% test substance concentration.

Skin reactions of grade 1 were observed in seven experimental animals in response to the 10% test substance concentration and in five experimental animals in response to the 5% concentration.

No skin reactions were evident in the control animals (see Table 4).

Table 4: SECOND CHALLENGE READINGS

	Animal No.	Day 24 Readings		Day 25 Readings		Comments
		10%^#	5%	10%	5%
Control	16	0	0	0	0
	17	0	0	0	0
	18	0	0	0	0
	19	0	0	0	0
	20	0	0	0	0
Experimental	21	1	1	0	0	sensitised ( 10%, 5%)
	22	0	0	0	0	not sensitised
	23	1	1	0	0	sensitised (10%, 5%)
	24	0	0	0	0	not sensitised
	25	1	0	0	0	sensitised (10%)
	26	1	1	0	0	sensitised (10%, 5%)
	27	1	1	1	0	sensitised (10%, 5%)
	28
	29	1	1	1	0	sensitised (10%, 5%)
	30	1	0	0	0	sensitised (10%)

#. Test substance concentration.

Toxicity/Mortality

One experimental animal was removed from the study on day 10 following the observation of emaciation, dark eyes and labored respiration. Macroscopic postmortem examination of the animal showed haemorrhages in the lungs. It was considered that the death of this animal was incidental and that the study outcome, based on the healthy surviving animals, was not adversely affected.

No further mortality occurred and no further symptoms of systemic toxicity were observed in the animals of the main study.

Body Weights

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period (see Table 5).

Table 5: BODY WEIGHTS (grams)

Group/Sex	Animal	Day 1	Day 32
Group 1/Females (Control)	16	315	470
	17	313	460
	18	310	469
	19	334	469
	20	337	514
	Mean	322	476
	St. Dev.	13	21
	N	5	5
Group 2/Females (Experimental)	21	324	510
	22	336	451
	23	319	497
	24	301	462
	25	342	518
	26	339	468
	27	322	528
	28	310	-
	29	342	487
	30	306	464
	Mean	324	487
	St. Dev.	15	28
	N	10	9

- Animal 28 was removed from the study on day 10 after showing signs of ill health.

Interpretation of results:

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

Conclusions:

No conclusions regarding the sensitisation rate could be drawn from the responses to the 20% concentration, since comparable reactions were seen in the experimental and control animals. The skin reactions observed in response to the 10% and 5% test substance concentrations in seven and five (of the nine) experimental animals in the challenge phase, respectively, were considered indicative of sensitisation, based on the absence of any response in the control animals to those concentrations.
These results indicate a sensitisation rate of 78 per cent to the 10% concentration and a sensitisation rate of 56 per cent to the 5% concentration.
Based on these results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC), TERT-BUTYLPEROXY ISOPROPYL CARBONATE, 75% SOLUTION IN AROMATIC FREE M INERAL SP IR IT should be labelled as: may cause sensitisation by skin contact (R 43).

Executive summary:

Assessment for Contact Hypersensitivity to TERT-BUTYLPEROXY ISOPROPYL CARBONATE, 75% SOLUTION IN AROMATIC FREE MINERAL SPIRIT in the Albino Guinea Pig (Maximisation Test).

The study was carried out based on the guidelines described in: EC Commission Directive 96/54/EC, Part 8.6, 'Skin Sensitisation' and OECD No. 406, "Skin Sensitisation", and based on the method described by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens.

Test substance concentrations selected for the Main study were based on the results of a preliminary study. In the Main study, experimental animals were intradermally injected with a 10% concentration and epidermally exposed to the undiluted test substance. Control animals were similarly treated, but with vehicle alone (corn oil). Two weeks after the epidermal application all animals were challenged with a 20% test substance concentration and the vehicle. A second challenge was performed one week later with the test substance concentrations 10% and 5%.

First Challenge: Skin reactions of grade 1 were observed in experimental and control animals in response to the 20% test substance concentration. Scaliness was seen in the treated skin site of one experimental animal.

Second challenge: Since comparable skin reactions were observed in both experimental and control animals, a second challenge was performed one week later. The animals were then treated with a 10% and a 5% test substance concentration. Skin reactions of grade 1 were observed in seven experimental animals in response to the 10% test substance concentration and in five experimental animals in response to the 5% concentration. No skin reactions were evident in the control animals.

One experimental animal was removed from the study on day 10 following the observation of emaciation, dark eyes and labored respiration. Macroscopic postmortem examination of the animal showed haemorrhages in the lungs. It was considered that the death of this animal was incidental and that the study outcome, based on the healthy surviving animals, was not adversely affected. No further mortality occurred and no further symptoms of systemic toxicity were observed in the animals of the main study.

No conclusions regarding the sensitisation rate could be drawn from the responses to the 20% concentration, since comparable reactions were seen in the experimental and control animals. The skin reactions observed in response to the 10% and 5% test substance concentrations in seven and five (of the nine) experimental animals in the challenge phase, respectively, were considered indicative of sensitisation, based on the absence of any response in the control animals to those concentrations. These results indicate a sensitisation rate of 78 per cent to the 10% concentration and a sensitisation rate of 56 per cent to the 5% concentration.

Based on these results and according to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Commission Directive 93/21/EEC), TERT-BUTYLPEROXY ISOPROPYL CARBONATE, 75% SOLUTION IN AROMATIC FREE MINERAL SPIRIT should be labelled as: may cause sensitisation by skin contact (R 43).

The substance is classified as a moderate skin sensitizer, Cat 1b per “Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures”, Version 5, July 2017 and per UN GHS.  It is considered as a moderate skin sensitizer (Guidance on information requirements and chemical safety assessment, Part E).

Reference 2

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The substance is a skin sensitizer, Cat 1B, based on in vivo testing (GPMT).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance is a skin sensitizer, Cat 1B, based on in vivo testing (GPMT).

The substance is classified as a moderate skin sensitizer, Cat 1B (H 317) per “Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures”, Version 5, July 2017 and per UN GHS.  It is considered as a moderate skin sensitizer (Guidance on information requirements and chemical safety assessment, Part E).