Reaction mass of 3,7-dimethyloct-6-en-1-yl 2-methylpropanoate and (2E)-3,7-dimethylocta-2,6-dien-1-yl 2-methylpropanoate and (2Z)-3,7-dimethylocta-2,6-dien-1-yl 2-methylpropanoate

Endpoint summary

Description of key information

Skin sensitisation potential is based on read across from Citronellyl butyrate which is tested in an LLNA type of test OECD TG 442B resulting in being a sensitiser 1B

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

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

Adequacy of study:

key study

Study period:

2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across information.

Justification for type of information:

The read across justification is presented in the Endpoint summary Skin Sensitisation. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

Key result

Parameter:

other: EC1.6 (%)

Value:

26.4

Key result

Parameter:

SI

Value:

1.22

Remarks on result:

other: test group: 1%

Key result

Parameter:

SI

Value:

1.1

Remarks on result:

other: Test group: 5%

Key result

Parameter:

SI

Value:

1.4

Remarks on result:

other: Test group: 10%

Key result

Parameter:

SI

Value:

1.78

Remarks on result:

other: Test group: 25%

Key result

Parameter:

SI

Value:

2.15

Remarks on result:

other: Test group: 50%

Key result

Parameter:

SI

Value:

1.7

Remarks on result:

other: Test group: 100%

Interpretation of results:

other: Skin sensitiser Category 1B

Remarks:

according to EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

The SI values for Geranyl Isobutyrate are considered the same as for Citronellyl butyrate: in a first set, at 25, 50 and 100%, were 1.78, 2.15 and 1.70, respectively. In a second set, at 1, 5 and 10%, SI values were 1.22, 1.10 and 1.40, respectively. These results show that the substance could elicit a SI ≥ 1.6. The EC1.6 is calculated to be of 26.4%. The substance is a skin sensitiser (Category 1B), based on the results of the source substance.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

(LLNA: BrdU)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 May 2016 - 22 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This information is used for Geranyl Isobutyrate MCS.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Species:

mouse

Strain:

other: CBA/N

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Japan SLC, Inc., Japan (producer); Central Lab. Animal Inc., Republic of Korea (Supplier)
- Females (if applicable) nulliparous and non-pregnant: not specified]
- Age at study initiation: 9 - 12 weeks
- Weight at study initiation: 18.5 - 21.0g (dose range finding); 17.6 - 21.7g (main set1); 19.1 - 25.7g (main set2)
- Housing: Group housing 2–3 animals/Polysulfone cage
- Diet: free access to Pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C)
- Water: free access to public tap water filtered and irradiated by ultraviolet light
- Acclimation period: 4 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 24.7
- Humidity (%): 44.5 - 65
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 31 May 2016 To: 21 December 2016

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 25, 50 and 100% (set 1); 0, 1, 5 and 10% (set 2)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: the test substance was dissolved in the vehicle in a preliminary solubility test.
- Two animals were observed per dose group. All mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded prior to dosing (Day 1) and on the day of necropsy, Day 6. Both ears of each mouse were observed for erythema and scored using erythema score. Ear thickness measurement was taken using a thickness gauge on Day 1 (predose), Day 3 and Day 6. Additionally, on Day 6, ear weight was determined by balance.

MAIN STUDY : the main study was conducted in two sets, with lower treatment doses used in the second set.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Following the quarantine-acclimation period (group assignment), 25 healthy females were selected and randomly distributed into 5 groups (5 females per group).
- Criteria used to consider a positive response: SI ≥ 1.6

TREATMENT PREPARATION AND ADMINISTRATION: A volume of 25 μL was applied to the dorsum of both ears of all animals daily for three consecutive days. The dose level of the positive substance was selected at 25%. Negative control animals were dosed with the vehicle, acetone/olive oil solution.

OBSERVATIONS:
- Clinical signs: All animals were observed for mortality, general condition and clinical signs for 6 days.
- Body weights: Body weights were recorded prior to dosing (day 1) and on the day of necropsy (day 6).
- Erythema: Both ears of each mouse were observed for erythema and scored for 6 days.
- Ear thickness: Ear thickness measurement was taken using a thickness gauge (543-681B, Mitutoyo Co., Japan) on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) and day 6 (the day of necropsy).

On day 5, a volume of 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected interperitoneally. Approximately 24 hours (24 h) after BrdU injection, the animals were euthanized under CO2 gas. The draining auricular lymph nodes from each mouse ear were excised and processed separately in phosphate buffered saline for each animal.

Preparation of cell suspension:
For each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally was prepared by #70 nylon mesh to generate a single cell suspension. In each case, the target volume of the LNC suspension was adjusted to the determined optimized volume. The optimized volume was based on the mean absorbance within 0.1-0.2 in the NC group.

Determination of cellular proliferation: BrdU was measured by ELISA: 100 μL of the LNC suspension was added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the LNC suspension, anti-BrdU antibody was added to each well and allowed to react. Subsequently, anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. Absorbance at 370 nm with a reference wavelength of 492 nm was measured.

BrdU labelling index = (ABSem - ABSblank,em) - (ABSref-ABSblank,ref)
with ABS = Absorption; em = emission wavelength; ref= reference substance

SI= mean of BrdU labelling index in the test substance / mean of BrdU labelling index in the negative control

Acceptability criterium: positive control should have a SI ≥ 1.6.

Evaluation criteria:
SI Result
SI < 1.6 Negative
SI ≥ 1.6 Positive

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical analysis was conducted using a statistical program (version 9.3, SAS Institute Inc., U.S.A.) for the data including body weight, erythema score, ear thickness, ear weight and stimulation index.

Bartlett’s test was employed on homogeneity of variance (significance level: 0.05) for body weights, ear thickness, ear weight and stimulation index data. One-way analysis of variance (ANOVA) was employed on homogeneous data. Dunnett’s t-test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group (G1, G6) and each of the test substance groups (G2–G4, G7–G9) or positive substance group (G5, G10). Since it was not significant, Kruskal-Wallis test was employed on heterogeneous data and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the
negative control group (G1, G6) and each of the test substance groups (G2–G4, G7–G9) or positive substance group (G5, G10).

Kruskal-Wallis test for the erythema score was employed on heterogeneous data, and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group (G1, G6) and each of the test substance groups (G2–G4, G7–G9) or positive substance group (G5, G10).

Positive control results:

Set 1: In the positive control group at 25%, the mean value of stimulation index was 3.10. There was a significant increase when compared to the negative control group (p<0.05).
Set 2: In the positive control group at 25%, the mean value of stimulation index was 4.32. There was a significant increase when compared to the negative control group (p<0.05).

Key result

Parameter:

other: EC1.6 (%)

Value:

26.4

Key result

Parameter:

SI

Value:

1.22

Remarks on result:

other: Test group: 1%

Key result

Parameter:

SI

Value:

1.1

Remarks on result:

other: Test group: 5%

Key result

Parameter:

SI

Value:

1.4

Remarks on result:

other: Test group: 10%

Key result

Parameter:

SI

Value:

1.78

Remarks on result:

other: Test group: 25%

Key result

Parameter:

SI

Value:

2.15

Remarks on result:

other: Test group: 50%

Key result

Parameter:

SI

Value:

1.7

Remarks on result:

other: Test group: 100%

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
- Set 1: In the negative control group, the mean value of stimulation index was 1.00. In the test substance groups at 25, 50 and 100%, the mean values of stimulation index were 1.78, 2.15 and 1.70, respectively. There were significant increases when compared to the negative control group (p<0.05: 25, 50 and 100%).
- Set 2: In the negative control group, the mean value of stimulation index was 1.00. In the test substance groups at 1, 5, 10%, the mean values of stimulation index were 1.22, 1.10 and 1.40, respectively. There were no significant differences when compared to the negative control group.

EC1.6 CALCULATION
Three concentration showed SI of <1.6, and EC1.6 was calculated to be 26.4%.

CLINICAL OBSERVATIONS: There were no abnormal clinical signs or deaths in any dosing group during the observation period in both experiments.

BODY WEIGHTS (mean)
- Set 1:
In the negative control group, the mean values of body weights were 19.3–20.2 g prior to dosing until Day 6 after dosing.
In the test substance groups at 25, 50 and 100%, the mean values of body weights were 19.1–19.5, 19.5–19.6 and 19.3–19.3 g, respectively. There were no significant differences when compared to the negative control group.
In the positive control group at 25%, the mean values of body weights were 19.6–19.9 g. There were no significant differences when compared to the negative control group.
- Set 2:
In the negative control group, the mean values of body weights were 21.1–21.3 g prior to dosing until Day 6 after dosing.
In the test substance groups at 1, 5, 10%, the mean values of body weights were 21.7–21.4, 21.6–20.6 and 21.9–20.8 g, respectively. There were no significant differences when compared to the negative control group.
In the positive control group at 25%, the mean values of body weight were 21.4–20.9 g. There were no significant differences when compared to the negative control group.

ERYTHEMA SCORE:
- Set 1:
In the negative control group, the mean values of erythema score at 0.0 from prior to dosing until Day 6 after dosing.
In the test substance groups at 25, 50 and 100%, the mean values of erythema score was 0.0–0.7, 0.0–0.9 and 0.0–1.2, respectively. There were significant increases when compared to the negative control group (p<0.05: Day 4 (25%), p<0.01: Days 4 (50 and 100%), 5 and 6 (25, 50 and 100%)).
In the positive control group at 25%, the mean values of erythema score was 0.0–1.9. There were significant increases when compared to the negative control group (p<0.01: Days 3, 4, 5 and 6).
- Set 2:
In the negative control group, the mean values of erythema score at 0.0 prior to dosing until Day 6 after dosing.
In the test substance groups at 1, 5, 10%, the mean values of erythema was 0.0, 0.0–1.1 and 0.0–1.0, respectively. There were significant increases when compared to the negative control group (p<0.01: Days 5 and 6 (5 and 10%)).
In the positive control group at 25%, the mean values of erythema score was 0.0–1.7. There was a significant increase when compared to the negative control group (p<0.01: Days 3, 4, 5 and 6).

EAR THICKNESS (mean):
- Set 1:
In the negative control group, the mean values of ear thickness at 0.19–0.20 mm prior to dosing until Day 6 after dosing.
In the test substance groups at 25, 50 and 100%, the mean values of ear thickness was 0.19–0.21, 0.20–0.21 and 0.19–0.21 mm, respectively. There were significant increases when compared to the negative control group (p<0.05: Day 6 (25%)).
In the positive control group at 25%, the mean value of ear thickness was 0.19–0.22 mm. There were significant increases when compared to the negative control group (p<0.01: Day 6).
- Set 2:
In the negative control group, the mean values of ear thickness at 0.19–0.19 mm prior to dosing until Day 6 after dosing.
In the test substance groups at 1, 5, 10%, the mean values of ear thickness were 0.20–0.19, 0.20–0.20, 0.20–0.20 mm, respectively. There was a significant increase when compared to the negative control group (p<0.05: Day 1 (1%), Day 3 (5%), p<0.01: Days 1 (5 and 10%), 3 (10%) and 6 (5 and 10%)).
In the positive control group at 25%, the mean value of ear thickness was 0.20–0.21 mm. There was a significant increase when compared to the negative control group (p<0.01: Days 1, 3 and 6).

EAR WEIGHTS (mean):
- Set 1:
In the negative control group, the mean value of ear weight was 12.6 mg.
In the test substance groups at 25, 50 and 100%, the mean values of ear weight were 13.5, 13.5 and 13.7 mg, respectively. There were significant increases when compared to the negative control group (p<0.05: 100%).
In the positive control group at 25%, the mean value of ear weight was 14.2 mg. There was a significant increase when compared to the negative control group (p<0.01).
- Set 2:
In the negative control group, the mean value of ear tissue was 12.1 mg.
In the test substance groups at 1, 5, 10%, the mean values of ear tissue were 12.9, 13.3 and 12.6 mg, respectively. There were significant increases when compared to the negative control group (p<0.05: 5%).
In the positive control group at 25%, the mean value of ear tissue was 13.9 mg. There was a significant increase when compared to the negative control group (p<0.01).

Interpretation of results:

other: Skin sensitiser Category 1B

Remarks:

according to EU CLP (EC No. 1272/2008 and its amendments).

Conclusions:

The substance produced a SI ≥ 1.6 and an EC1.6 value of 26.4% was calculated. Based on these results, the substance is considered to be a skin sensitiser.

Executive summary:

In a Local Lymph Node Assay (BrdU-ELISA), the skin sensitisation potential of the substance was tested according to OECD TG 442B under GLP. Per concentration, 5 female mice were used. Skin sensitization potential was evaluated by ear thickness measurements. Cellular proliferations and SI were determined. In the first set, at 25, 50 and 100%, SI values were 1.78, 2.15 and 1.70, respectively. In the second set, at 1, 5 and 10%, SI values were 1.22, 1.10 and 1.40, respectively. Negative and positive controls were included and all acceptability criteria were met (SI values positive controls: 3.10 and 4.32). These results show that the substance could elicit a SI ≥ 1.6. An EC1.6 value of 26.4% was calculated. Based on the results, the substance is considered a skin sensitiser 1B.

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisation potential of Geranyl Isobutyrate MCS is based on read-across from Citronellyl butyrate. The executive summary of the skin sensitisation results of the latter are presented below and are followed by the read-across rationale.

Skin sensitisation information from Citronellyl butyrate

In a Local Lymph Node Assay (BrdU-ELISA), the skin sensitisation potential of the substance was tested according to OECD TG 442B test guideline and GLP principles. Per concentration, 5 female mice were used. Skin sensitization potential was evaluated by ear thickness measurements. Cellular proliferations and SI were determined. In the first set, at 25, 50 and 100%, SI values were 1.78, 2.15 and 1.70, respectively. In the second set, at 1, 5 and 10%, SI values were 1.22, 1.10 and 1.40, respectively. Negative and positive controls were included and all acceptability criteria were met (SI values positive controls: 3.10 and 4.32). These results show that the substance could elicit a SI ≥ 1.6. An EC1.6 value of 26.4% was calculated. Based on the results, the substance is considered a skin sensitiser.

The skin sensitizing potential of Geranyl Isobutyrate MCS based on read across from data available for Citronellyl butyrate (CAS #141-16-2)

 

Introduction and hypothesis for the analogue approach

Geranyl Isobutyrate MCS consists of 4 constituents which areIsobutyrate esters of a 3,7-dimethyloctanol chain and can be divided into two subgroups based on the number of unsaturated bonds. The Geranyl-type subgroup consists of two isomers of Geranyl Isobutyrate MCS, which have two double bonds in the chain and are present at a total of 70%. The Citronellyl-type subgroup consists of Citronellyl Isobutyrate, which has one double bond in the chain and is present at ca. 25% and a Citronellyl like component without a double bond which is present at <5%.

For Geranyl Isobutyrate MCS there are no skin sensitisation data available.In accordance with Article 13 of REACH, lacking information can be generated whenever possible by i.e. applying alternative methods such as grouping and read-across.For assessing the skin sensitisation potential of Geranyl Isobutyrate MCS, the analogue approach is selected because for structural analogue, Citronellyl butyrate, skin sensitisation data is available which can be used for read across.

Hypothesis: Geranyl Isobutyrate has the same skin sensitisation potential asCitronellyl butyrate.

Available information:For Citronellyl butyrate an LLNA type of study was performed according to OECD TG 442B (Rel. 1) Dose levels selected were 100, 50, 25, 10 and 5% and 2 mice were used per dose group. Both ears of each mouse were scored for erythema, and ear thickness and ear weight were determined. The EC1.6 (the relevant cut off for this test) was calculated to be 26.4%. Based on these results Citronellyl butyrate is classified as sensitizing cat. 1B.

Target chemical and source chemical(s)

Chemical structures of the target chemical and the source chemical(s) are shown in the data matrix, including physico-chemical properties and available toxicologicalinformation. Furthermore, a full list of constituents of Geranyl Isobutyrate MCS, including information relevant for read-across, is given in Appendix 1.

Purity / Impurities

The unidentified impurities of Geranyl Isobutyrate MCS are not considered to have a significant influence on the skin sensitising potential in bacteria.

Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and a common functional group. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation.

Analogue selection: For Geranyl Isobutyrate (MCS)Citronellyl butyrate was selected as an analogue because the constituents of Geranyl Isobutyrate MCS share a similar backbone and the same functional groups and for Citronellyl butyrated LLNA type of information is available.

Structural similarities and differences: Geranyl Isobutyrate MCS and Citronellyl butyrate havethe same 3,7-dimethyloctanol chain backbone and a butyl ester as a functional group. The difference is that Geranyl Isobutyrate MCS has two double bonds in this chain, while Citronellyl butyrate has one double bond. Also the Geranyl Isobutyrate MCS has an Isobutyl chain versus a straight butyl chain.

Bioavailability: The dermal bioavailability is very similar for Geranyl Isobutyrate MCS and Citronellyl butyrate based on the same the molecular weight and the physico-chemical properties such as log Kow. The log Kow of both substances and the constituents are around 5.5.

Reactivity: The reactivity of Geranyl Isobutyrate MCS versus Citronellyl butyrate is considered similar in view of the similar functional terpene-type of groups (double bonds with a methyl group attached), which after auto-oxidation, can present skin sensitizing properties.

Uncertainty of the prediction:Some of Geranyl Isobutyrate MCS constituents have a conjugated double bond with the ester group which is not present in Citronellyl butyrate. This may present a slightly higher reactivity. In view of the limited dermal absorption this slightly higher reactivity is not expected to change significantly the skin sensitisation potential. There are no remaining uncertainties other than those presented.

Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the data matrix below.

                                  

Conclusions for hazard and risk assessment

For Geranyl Isobutyrate MCS there is no experimental skin sensitisation information available. Citronellyl butyrate is a suitable analogue which is used for read across. When using read across the result derived should be applicable for C&L and/or risk assessment and be presented with adequate and reliable documentation. This documentation is presented in the current document. For Citronellyl butyrate a positive LLNA:BrdU-ELISA (OECD TG 442B) is available with anEC1.6 of 26.4% and this information will be directly used for Geranyl Isobutyrate MCS.

Final conclusion on hazard and risk assessment: Geranyl Isobutyrate MCS is a skin sensitizer 1B and has an EC1.6 of 26.4%.

 

Data matrix for Geranyl Isobutyrate MCS and its analogue Citronellyl butyrate

Common name	Geranyl Isobutyrate MCS	Citronellyl butyrate
	Target	Source
Chemical name	n.a.	3,7-dimethyloct-6-en-1-yl butanoate
Chemical structures	For a full list of constituents, see Appendix 1.
CAS #	--	141-16-2
EC #	905-357-4	205-463-4
REACH registered	2018	Registered
Empirical formula	n.a.	C14H26O2
SMILES	n.a.	CCCC(=O)OCCC(C)CCC=C(C)C
Physico-chemical data
Molecular weight	n.a.	226
Physical state	Liquid	Liquid
Log Kow	5.7 (exp.)	5.54 (est.)
Ws (mg/L)	17.4 (exp.)	1.63 (est.)
Vp (Pa)	1.2 (exp.)	6.2 (est.)
Human health endpoints
Skin sensitisation	Read across: Skin Sens. Cat. 1B	Skin Sens. Cat. 1B (EC1.6: 26.4%) (OECD 442B)

 

Appendix 1. Data matrix for the constituents of Geranyl Isobutyrate MCS and Citronellyl butyrate

Common names	Geranyl Isobutyrate MCS				Citronellyl butyrate
	1	2	3	4
Constituents	3,7-dimethyloctyl 2-methylpropanoate	3,7-dimethyloct-6-en-1-yl 2-methylpropanoate	(2Z)-3,7-dimethylocta-2,6-dien-1-yl 2-methylpropanoate	(2E)-3,7-dimethylocta-2,6-dien-1-yl 2-methylpropanoate	3,7-dimethyloct-6-en-1-yl butanoate
Chemical structures
CAS no	71662-25-4	97-89-2	2345-24-6	2345-26-8	141-16-2
Concentration range	3.7	25	14	56
Empirical formula	C14H28O2	C14H26O2	C14H24O2	C14H24O2	C14H26O2
Molecular weight	228	226	224	224	226
Physico-chemical data*
Water solubility, mg/l	0.56	0.68	0.82	0.82	0.59
Log Kow	5.6	5.5	5.4	5.4	5.5

* Episuite v4.11

Justification for classification or non-classification

Based on the results, the substance is considered a skin sensitiser 1B and shall be labelled with H317: May cause an allergic skin reaction according to EU CLP (EC No. 1272/2008 and its amendments).