Reaction mass of 6,6-dimethyl-2-methylidenebicyclo[3.1.1]heptane and rel-6,6-dimethylspiro[bicyclo[3.1.1]heptane-2,2'-oxirane]

Endpoint summary

Description of key information

Skin sensitisation: sensitising (sensitizer 1B), based on read-across from Prismantol, which was tested in OECD TG 406.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

GPMT test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 17 September and 12 October 1991.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

The information is used for read across to Rosmarel. The GPMT test with Prismantol was performed before LLNA or in vitro tests became the first test of selection.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

(1981)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The GPMT test was performed before LLNA or in vitro tests became the first test of selection.

Specific details on test material used for the study:

The test substance was gently melted in a water bath at 60°C and prepared prior to each application in Alembicol D.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England
- Sex: females, nulliparous and non-pregnant
- Age at Acclimatization Start: ap. 6 to 7 weeks of age
- Weight at Acclimatization Start: 304 to 353 g
- Housing: in groups of ten in suspended metal cages with wire mesh floors.
- Diet: a vitamin C-enriched guinea-pig Diet F.D.1. and drinking water were provided ad libitum. Hay was given weekly to provide dietary supplement. The routine analyses of diet and water was done.
- Water: free access to tap water
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal

Vehicle:

other: Alembicol D

Remarks:

Supplied by Alembic Products, Saltney, Chester, England

Concentration / amount:

7.5%

Route:

epicutaneous, occlusive

Vehicle:

other: Alembicol D

Remarks:

Supplied by Alembic Products, Saltney, Chester, England

Concentration / amount:

60%

Day(s)/duration:

48 h

Route:

epicutaneous, occlusive

Vehicle:

other: Alembicol D

Remarks:

Supplied by Alembic Products, Saltney, Chester, England

Concentration / amount:

25 and 50%

Day(s)/duration:

24 h

No. of animals per dose:

Test animals: 20
Control animals: 10

Details on study design:

RANGE FINDING TESTS
- Intradermal injections:
Intradermal injections were made into the clipped flank of animals at concentrations of 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0% of the test substance in Alembicol D. The reactions were examined after 24 and 72 hours for size, erythema and oedema.
As well-defined erythema (grade 2) was observed with 7.5% in both animals, this concentration was selected as the intradermal induction concentration.

- Epidermal application:
Patches of filter paper (2x4 cm) were saturated with 0.4 ml of the undiluted test substance at the concentration of 60% in Alembicol D. These were applied to the clipped and shaved flanks of each of 2 males and 2 females and were covered by a length of impermeable plastic adhesive tape. This was firmly secured by elastic adhesive bandage wrapped around the trunk and fixed with "Sleek" impervious plastic adhesive tape. The patches were removed 48 hours after application and the treatment sites were examined immediately and 24 hours and 48 hours after removal of the patches.
At 60% in three animals only very slight erythema (grade 1) was observed immediately after patch removal and in three animals after 24 and 48 hours, in one animal a well-defined erythema (grade 2) was observed after 24 and 48 hours, and in one animal a necrotic patch was observed. Therefore, this concentration was used for the topical induction concentration.
As at 50% no erythema was observed at any time of observation, this concentration was used for the highest challenge exposure.

MAIN STUDY
A. INDUCTION EXPOSURE
1) Intradermal injections (0.1 ml)
- Concentration: 7.5%
- Site: the dorsal scapular region
Three pairs of intradermal injections:
1) Freund's complete adjuvant (FCA) 50:50 with water for irrigation
2) Test substance at 7.5% in Alembicol D
3) Test substance at 7.5% in Alembicol D in a 50:50 mixture of FCA with Alembicol D
Control group:
1) Freund's complete adjuvant (FCA) 50:50 with water for irrigation
2) Alembicol D
3) 50:50 mixture of FCA with water for irrigation
- Readings: daily
- Results: Erythema and edema was observed in both groups

2) Topical applications one week after the injections:
- Concentration: 60%
- Amount: saturated patch (control animals: water for irrigation only)
- Area: 8 cm2
- Exposure period: 48 hours (occlusive)
- Readings: immediately after patch removal and 24 and 48 hours after patch removal

B. CHALLENGE EXPOSURE (control and test group)
- Day of challenge: 2 weeks after the epidermal induction application
- Concentrations: 25 and 50%
- Exposure period: 24 hours (occlusive)
- Sites: left flank, anterior and posterior sites
- Amount: saturated patch
- Readings: at 24, 48 and 72 hours after patch removal

Positive control substance(s):

yes

Remarks:

Formalin

Positive control results:

The results of the latest sensitivity check of August-September 1991 showed that the system was responsive (10 of 10 animals reacted positive).

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Localized dermal reactions, dryness and sloughing of the epidermis

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

19

Total no. in group:

20

Clinical observations:

Localized dermal reactions, dryness and sloughing of the epidermis

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

5% and 1% formalin

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive 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:

10

Remarks on result:

no indication of skin sensitisation

Preliminary study:

- At topical application of 60% no edema was observed at any time point and in three animals a very slight erythema (grade 1) was observed immediately after patch removal and in all four exposed animals after 24 and 48 hours, of which in one animal a well-defined erythema (grade 2) was observed after 24 and 48 hours, and in one animal a necrotic patch was observed. Therefore, this concentration was used for the topical induction concentration. As at 50% no erythema was observed at any time of observation, this concentration was used for the highest challenge exposure.

Main study:

- The body weight gain of the animals was normal, no signs of ill health, no systemic toxicity and no mortality was seen.

- The ten animals exposed to negative control did not show any signs of irritation.

- Intradermal injections: Application area around the injection sites (at 7.5%) was found to show a slight irritation.

Interpretation of results:

other: Skin sensitizer Category 1B

Remarks:

According to EU CLP (EC/1272/2008 and its amendments).

Conclusions:

In a guinea pig maximisation test performed similar to OECD 406 (1981) and according to GLP principles, the substance is considered a 1B skin sensitiser.

Executive summary:

The skin sensitisation potential of Prismantol was tested in a guinea pig maximisation test performed similar to OECD 406 (1981) and according to GLP principles. A concentration of 7.5% was used for the intradermal induction, 60% for the epidermal induction and 25 and 50% for the topical challenge. The substance produced evidence of skin sensitization in all tested twenty animals and therefore was considered a skin sensitiser. Because ≥ 30% (100%) of animals responded at a dose of > 1% (7.5%), Prismantol is 1B sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Remarks:

GPMT test

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across information

Justification for type of information:

The full read-across document can be found in the Endpoint Summary in text and in the attached file.

Reason / purpose for cross-reference:

read-across source

Justification for non-LLNA method:

The GPMT test was performed before LLNA or in vitro tests became the first test of selection.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

20

Total no. in group:

20

Clinical observations:

Localized dermal reactions, dryness and sloughing of the epidermis

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25%

No. with + reactions:

19

Total no. in group:

20

Clinical observations:

Localized dermal reactions, dryness and sloughing of the epidermis

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

5% and 1% formalin

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive 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:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

other: Skin sensitizer Category 1B

Remarks:

According to EU CLP (EC/1272/2008 and its amendments).

Conclusions:

Skin sensitisation: sensitising (sensitizer 1B), based on read-across from Prismantol, which was tested in OECD TG 406.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

For assessing the skin sensitization of Rosemarel, read across from Prismantol (CAS# 122760-84-3) is used. First the data of Prismantol will be presented and thereafter the read across justification. For Rosemarel also a HRIPT study is available, which cannot be used to assess the skin sensitization potential sufficiently, in accordance with REACH.

Skin sensitization with Prismantol:

The skin sensitisation potential of Prismantol was tested in a guinea pig maximisation test performed similar to OECD 406 (1981) and according to GLP principles. A concentration of 7.5% was used for the intradermal induction, 60% for the epidermal induction and 25 and 50% for the topical challenge. The substance produced evidence of skin sensitization in all tested twenty animals and therefore was considered a skin sensitiser. Because ≥ 30% (100%) of animals responded at a dose of > 1% (7.5%), Prismantol is 1B sensitizer.

Rosemarel and its sensitising potential using read across from Prismantol (CAS 122760-84-3)

 

Introduction and hypothesis for the analogue approach

Rosemarel is a multi-constituent. The major part is the constituent Beta-pinene (51%) and this constituent has a hexyl ring bridged with one carbon, to which two methyl groups are attached. At the alpha position there is a double bonded methyl group attached to the hexyl ring, which is the functional group. The minor constituent (36%) has the same backbone. The functional group is an epoxide in the place where there is the double bonded methyl group in the other component. For Rosemarel limited skin sensitisation data are available, which are not sufficient to make a decision for this endpoint. Therefore, additional information is used in accordance with Article 13 of REACH where it is said that lacking information should be generated whenever possible by means other than vertebrate animal tests, i.e. applying alternative methods such as in vitro tests, SARs, grouping and read-across.

Hypothesis: Rosemarel is expected to have the same sensitising properties and potency as Prismantol.

Available experimental information: For the target substance, Rosemarel, only one HRIPT is available, which, according to the REACH guidance, cannot cover the skin sensitisation endpoint. For the source chemical, Prismantol, a well conducted GPMT is available showing a sensitization potential (IFF, 1992; GLP, according to OECD 406, K1). In this study, the intradermal induction was done at 7.5% and the topical induction at 60%, and the topical challenge was done at 50 and 25%. All 20 animals had a positive reaction at 25%. Therefore, Prismantol is a weak sensitizer 1B (≥ 30% of animals responding at a dose > 1%). This study with Prismantol will be used for read-across.

 

Target and Source chemical(s):

The target is Rosemarel, and the source is Prismantol. The information on the target and source substances, together with their physico-chemical properties, is presented in the data matrix below.

 

Purity / Impurities:

Rosemarel consists of Beta-pinene (51 %) and its oxidised form- the epoxide (36 %). The impurities are all below < 10%. The purity of the source Prismantol is reported to be 97%.

 

Analogue justification

According to REACH Annex XI, an analogue approach and structural alert information can be used to replace testing when information from different sources provides sufficient evidence to conclude that this substance has or does not have a particular dangerous property. The result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation.

Analogue selection:

First, the information on Beta-pinene was assessed, for which skin sensitisation information is available. Whether this would cover also Rosemarel’s epoxide constituent was questioned at first. Beta-pinene however is not expected to be a skin sensitizer and using Beta-pinene as an analogue would not account for the epoxide component of Rosemarel. Therefore, instead of Beta-pinene, IFF prefers to use Prismantol from its own portfolio because this substance also has an alcohol as a functional group and potentially reflects some similar reactivity as Rosemarel’s epoxide.

Structural analogy:

The main constituent of Rosemarel, Beta-pinene, has a very similar structure as Prismantol. Both substances have the same hexyl backbone being bridged with one or more carbons. They both have a carbon attached to this ring with a double bond. Beta-pinene has no other functional groups while Prismantol has an alcohol attached to the hexyl ring. Rosemarel’s epoxide has this group at the site of the double bond. An epoxide is more reactive compared to an alcohol.

Bioavailability:

Rosemarel (both constituents) has similar molecular weight, log Kow and water solubility as Prismantol, indicating similar dermal bioavailability. The difference in appearance between Rosemarel and Prismantol, liquid versus solid, is unlikely to present a difference in reactivity. 

Reactivity:

Rosemarel’s Beta-pinene constituent will be oxidized in the skin and form an epoxide causing similar skin sensitisation potential. Also Prismantol will become oxidized in skin and becomes an epoxide, which makes it very similar to Rosemarel once in skin. Therefore, similar reactivity between Rosemarel and Prismantol is anticipated. 

Remaining uncertainties:

There are no remaining uncertainties. Rosemarel’s Beta-pinene constituent has a similar potency as Prismantol. Rosemarel’s epoxide is expected to have a similar potency compared to the oxidised Beta-pinene and therefore Prismantol can be used for read across.

 

Conclusions per endpoint for C&L and risk assessment

For Rosemarel insufficient skin sensitisation information is available and the analogue Prismantol information on skin sensitisation can be used for read across. Prismantol is sensitizing in a GPMT test (OECD TG 406) in all twenty tested animals. The positive reactions were seen in ≥ 30% of animals (100%) after application of a dose > 1% (7.5%), it is a sensitizer 1B. Based on read across from Prismantol, Rosemarel is expected to be a skin sensitizer 1B.

 

Data matrix information on Rosemarel and Prismantol important for assessment of skin sensitising properties

Name of substance	Rosemarel	Prismantol	Beta-pinene
	Target	Source	Supporting (LLNA is available)
Chemical structure
Empirical	C10H16 and C10H16O	C12H18O	C10H16
Cas No	127-91-3 and 6931-54-0, respectively	122760-84-3	127-91-3
REACH registration	To be registered for 2018	NONS	Registered (2010)
EINECS	-	406-330-5	242-060-2
Mol weight	136.24 and 152.24	178.28	136.24
Phys-chem	EpiSuite predictions (C) and IFF measured (for both constituents)	EpiSuite predictions (C) and IFF measured
Appearance	Colourless to slight yellow liquid (IFF, 2016)	White crystal
Melting point (^oC)	<-20 (IFF, 2016)	50.74 (C) 42.5 - 49°C (IFF)
Boiling point (^oC)	173.85 (C) 172.2 (IFF, 2016)	258.98 (C)
Vapour pressure (Pa at 25^oC)	345.9 (IFF, 2016)	0.14 (C) 0.5 (IFF)
Water solubility (mg/l)	7.1 and 338 (C) 45.7 (IFF, 2016)	318.6 (C) 1266 (IFF)
Log Kow	4.35 and 2.95 (C) 3.2-3.4 (IFF, 2016)	3.23 (C) 2.85 (IFF)
Human health
Skin sensitization	Read across from Prismantol	Sensitizer in GPMT (OECD TG 406); Negative in LLNA up to 30% (OECD TG 429)	Positive in LLNA at 29% (OECD TG 429)

C: calculated value, the physico-chemical data were generated with EPISuite (version 4.1).

Justification for classification or non-classification

Rosemarel is a skin sensitizer based on read across from Prismantol, which was tested in a GPMT test. Based on fact that ≥ 30% (100%) of animals responded at a dose > 1% (7.5%) of Prismantol, Rosemarel has to be classified for skin sensitisation, according to EU CLP (EC1272/2008 and its amendments): Category 1B, H317: May cause an allergic skin reaction.