O-isobutyl ethylthiocarbamate

Endpoint summary

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of O-isobutyl ethylthiocarbamate (IBETC). Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of O-isobutyl ethylthiocarbamate (IBETC).

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS: Mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks (beginning of treatment)
- Weight at study initiation: 18.3 - 23.3 g
- Housing:single caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst / Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf), ad libitum
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg)
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): relative humidity 45-65%
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.

Vehicle:

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

Concentration:

The highest test item concentration which can be technically used was 100% of the undiluted test item. The dilutions were formulated in acetone/olive oil (4:1 v/v).
In the pre-test, two mice were treated with test item concentrations of 50 or 100%.
The test item in the main study was assayed at 25, 50, and 100%.

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration which can be technically used was 100% of the undiluted test item. The dilutions were formulated in acetone/olive oil (4:1 v/v).
In the pre-test, two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 50 or 100% once daily each on three consecutive days. At those concentrations the animals did not show signs of systemic toxicity or local irritation.

MAIN STUDY:

TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 25, 50, and 100% (w/v) in acetone:olive oil (4:1). The application volume, 25 µl, was spread over the entire dorsal surface (~ 8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
ADMINISTRATION OF ³H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED ³H-METHYL THYMIDINE

Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after treatment with ³HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a ß-scintillation counter.

INTERPRETATION OF RAW DATA

The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of ³HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
OBSERVATIONS

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
- Mortality / Viability: Once daily (week day) from experimental start to necropsy.
- Body weights: In the pre-test prior to the first application and prior to sacrifice; in the main experiment prior to the first application and prior to treatment with 3HTdR.
- Clinical signs (local / systemic): In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. In the main experiment clinical signs were recorded within 1 hour each application, and 24 ± 4 hours after the first and second application as well as on the day of preparation. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Positive control results:

Experiment performend in November 2009:
5, 10, and 25% alpha-hexyl cinnamic aldehyde in acetone:olibe oil (4:1) yielded a S.I. of 1.78, 2.54, and 4.88, respectively. The EC3 value calculated was 12.9%.
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

In this study Stimulation Indices of 1.22, 0.99, and 3.46 were determined with the test item at concentrations of 25, 50, and 100% in acetone/olive oil (4:1 v/v), respectively. A conventional dose response curve could not be established and an EC3 value was not calculated because of the odd dose response.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: As depicted in the table below.

Calculation and results of individual data; Vehicle: acetone/olive oil (4:1 v/v)

Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b )	S.I.
---	BG I	17	---	---	---	---
---	BG II	17	---	---	---	---
---	1	3517	3500	8	437.5	---
25	2	4282	4265	8	533.1	1.22
50	3	3479	3462	8	432.8	0.99
100	4	12129	12112	8	1514.0	3.46

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

 

Interpretation of results:

other: not sensitising

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Based on the numerical values of the stimulation indices one of which is slightly above 3, the test item Carbon disulfide has to be considered a skin sensitizer under the test condidions of this study. However, a conventional dose-response curve could not be established. An EC3 value was not calculated because of the odd dose-response.These factors contribute also in determining whether a borderline result shall be deemed as positive. In the opinion of the applicant this is a false positive and CS2 cannot be considered as a skin sensitizer base don this study.
Carbon disulphide (CAS number 75–15–0) is both reagents used in the manufacture of O-isobutyl ethylthiocarbamate (IBETC). Therefore, Carbon disulphide (CAS number 75–15–0) need to be considered in the assessment of O-isobutyl ethylthiocarbamate (IBETC).

Executive summary:

In this study carbon disulfide dissolved in acetone/olive oil (4:1 v/v) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay according to OECD guideline 429 was performed using test item concentrations of 25, 50, and 100%. All treated animals survived the scheduled study period and no signs of systemic toxicity or local irritation were observed. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration results in a 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices of 1.22, 0.99, and 3.46 were determined with the test item at concentrations of 25, 50, and 100% in acetone/olive oil (4:1). Based on the numerical values of these stimulation indices, the test item carbon disulfide has to be considered a skin sensitizer under the test conditions of this study. However, a conventional dose-response curve could not be established. An EC3 value was not calculated because of the odd dose-response. These factors contribute also in determining whether a borderline result shall be deemed as positive. In the opinion of the applicant this is a false positive and CS2 cannot be considered as a skin sensitizer base don this study.

Reference 2

Endpoint:

skin sensitisation: in chemico

Data waiving:

other justification

Justification for data waiving:

other:

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Propan-2-ol (Isopropyl alcohol) is a very close analogue of isobutyl alcohol which is used in the manufacture of O-isobutyl ethylthiocarbamate (IBETC).Therefore, the health effects of isopropyl alcohol need to be considered in the assessment of O-isobutyl ethylthiocarbamate (IBETC) .

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

cclimation perios is 4 days instead of 5 days.Pre and post study body weight not mentioned in the report

GLP compliance:

not specified

Type of study:

Buehler test

Justification for non-LLNA method:

This test determined the potential of the test substance to be sensitizing to skin.
20 animals in the test substance group and 10 animals in the vehicle control were treated with 100% Isopropyl alcohol
Four animals were exposed for 6 hours period to various concentrations of the test substance

Specific details on test material used for the study:

- Name of test material (as cited in study report): Isopropyl Alcohol (TSIN-Y0370-01)
EC Name: propan-2-ol
CAS Number :67-63-0
EC Number:200-661-7
CAS Name: isopropyl alcohol
- Molecular formula (if other than submission substance): (CH3)2 CHOH
- Molecular weight (if other than submission substance): 60.10
- Substance type: Pure active substance
- Physical state: liquid
- Analytical purity: Not available
- Impurities (identity and concentrations): None
- Composition of test material, percentage of components: 100% Isopropyl alchohol
- Isomers composition: None
- Purity test date: Not available
- Lot/batch No.: See below
- Expiration date of the lot/batch: See below
- Stability under test conditions: Not available
- Storage condition of test material: Room temperature

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Murphy Breeding Laboratories
- Age at study initiation: Not available
- Weight at study initiation: Not available
- Housing: housed singly in wire mesh cages
- Diet (e.g. ad libitum): Purina Laboratory Guinea Pig chow , ad libitum
- Water (e.g. ad libitum): animals were maintained on medicated water containing 4% of sulfaethoxypyridazine for four days and after that they were
furnished with non-medicated water, ad libitum
- Acclimation period: atleast 4 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not available
- Humidity (%): Not available
- Air changes (per hr): Not available
- Photoperiod (hrs dark / hrs light): 12 hour light /12 hour dark cycle


IN-LIFE DATES: From: 1980-08-08 To: 1980-09-06

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

One group of 20 test animals was treated with 0.4ml of 100% Isopropyl alcohol for a period of 6 hours weekly for 3 induction exposure.
Test group and 10 control animals were challenged with 100% Isopropyl alcohol.

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

One group of 20 test animals was treated with 0.4ml of 100% Isopropyl alcohol for a period of 6 hours weekly for 3 induction exposure.
Test group and 10 control animals were challenged with 100% Isopropyl alcohol.

No. of animals per dose:

20 animals in the test substance group and 10 animals in the vehicle control were treated with 100% Isopropyl alcohol

Details on study design:

RANGE FINDING TESTS: Four animals were exposed for 6 hours period to various concentrations of the test substance
Concentration: 100%, 50%, 25%, 10% v/v solution in distilled water
Exposure period: 24 hours
Grading: the patch site were scored for irritation four to five hours later after washing



MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 induction exposure
- Exposure period: 6 hours
- Test groups: 0.4 ml of undiluted test substance
- Control group: none
- Site: upper left quadrant of the backs of the test animals
- Frequency of applications: once a week for 3 weeks
- Duration: 6 hours webril patches under occlusion
- Concentrations: 0.4 ml of undiluted test substance


B. CHALLENGE EXPOSURE
- No. of exposures: one 6 hour exposure
- Day(s) of challenge: two weeks after second induction
- Exposure period: 6 hours under occlusion
- Test groups: 0.4 ml of undiluted test substance
- Control group: 0.4 ml of undiluted test substance
- Site: lower left quedrant of the back of the test animals
- Concentrations: 0.4 ml of undiluted test substance
- Evaluation (hr after challenge): 24 and 48 hours after challenge exposure

OTHER: None

Challenge controls:

10 animals treated with 100% Isopropyl alcohol

Positive control substance(s):

no

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100% Isopropyl alcohol

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no animals with positive responses

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100% Isopropyl alcohol. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no animals with positive responses.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100% Isopropyl alcohol

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no animals with positive responses

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100% Isopropyl alcohol. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no animals with positive responses.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100% Isopropyl alcohol

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no animals with positive responses

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100% Isopropyl alcohol. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no animals with positive responses.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100% Isopropyl alcohol

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no animals with positive responses

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100% Isopropyl alcohol. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no animals with positive responses.

Interpretation of results:

not sensitising

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Substance did not induce sensitization in the guinea pig model.
Propan-2-ol (Isopropyl alcohol) is a very close analogue of isobutyl alcohol which is used in the manufacture of O-isobutyl ethylthiocarbamate (IBETC).Therefore, the health effects of isopropyl alcohol need to be considered in the assessment of O-isobutyl ethylthiocarbamate (IBETC).

Executive summary:

One group of 20 test animals was treated with undiluted Isopropyl alcohol for a period of 6 hours weekly for 3 induction exposures. The test animals and control animals were challenged with undiluted Isopropyl alcohol. No skin reactions were observed in the test and control animals therefore, it was concluded that Isopropyl alcohol is not a sensitizer.

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

cclimation perios is 4 days instead of 5 days.Pre and post study body weight not mentioned in the report

GLP compliance:

not specified

Type of study:

Buehler test

Justification for non-LLNA method:

This test determined the potential of the test substance to be sensitizing to skin.
20 animals in the test substance group and 10 animals in the vehicle control were treated with 100% Isopropyl alcohol
Four animals were exposed for 6 hours period to various concentrations of the test substance

Specific details on test material used for the study:

- Name of test material (as cited in study report): Isopropyl Alcohol (TSIN-Y0370-01)
EC Name: propan-2-ol
CAS Number :67-63-0
EC Number:200-661-7
CAS Name: isopropyl alcohol
- Molecular formula (if other than submission substance): (CH3)2 CHOH
- Molecular weight (if other than submission substance): 60.10
- Substance type: Pure active substance
- Physical state: liquid
- Analytical purity: Not available
- Impurities (identity and concentrations): None
- Composition of test material, percentage of components: 100% Isopropyl alchohol
- Isomers composition: None
- Purity test date: Not available
- Lot/batch No.: See below
- Expiration date of the lot/batch: See below
- Stability under test conditions: Not available
- Storage condition of test material: Room temperature

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Murphy Breeding Laboratories
- Age at study initiation: Not available
- Weight at study initiation: Not available
- Housing: housed singly in wire mesh cages
- Diet (e.g. ad libitum): Purina Laboratory Guinea Pig chow , ad libitum
- Water (e.g. ad libitum): animals were maintained on medicated water containing 4% of sulfaethoxypyridazine for four days and after that they were
furnished with non-medicated water, ad libitum
- Acclimation period: atleast 4 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): Not available
- Humidity (%): Not available
- Air changes (per hr): Not available
- Photoperiod (hrs dark / hrs light): 12 hour light /12 hour dark cycle


IN-LIFE DATES: From: 1980-08-08 To: 1980-09-06

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

One group of 20 test animals was treated with 0.4ml of 100% Isopropyl alcohol for a period of 6 hours weekly for 3 induction exposure.
Test group and 10 control animals were challenged with 100% Isopropyl alcohol.

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

One group of 20 test animals was treated with 0.4ml of 100% Isopropyl alcohol for a period of 6 hours weekly for 3 induction exposure.
Test group and 10 control animals were challenged with 100% Isopropyl alcohol.

No. of animals per dose:

20 animals in the test substance group and 10 animals in the vehicle control were treated with 100% Isopropyl alcohol

Details on study design:

RANGE FINDING TESTS: Four animals were exposed for 6 hours period to various concentrations of the test substance
Concentration: 100%, 50%, 25%, 10% v/v solution in distilled water
Exposure period: 24 hours
Grading: the patch site were scored for irritation four to five hours later after washing



MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3 induction exposure
- Exposure period: 6 hours
- Test groups: 0.4 ml of undiluted test substance
- Control group: none
- Site: upper left quadrant of the backs of the test animals
- Frequency of applications: once a week for 3 weeks
- Duration: 6 hours webril patches under occlusion
- Concentrations: 0.4 ml of undiluted test substance


B. CHALLENGE EXPOSURE
- No. of exposures: one 6 hour exposure
- Day(s) of challenge: two weeks after second induction
- Exposure period: 6 hours under occlusion
- Test groups: 0.4 ml of undiluted test substance
- Control group: 0.4 ml of undiluted test substance
- Site: lower left quedrant of the back of the test animals
- Concentrations: 0.4 ml of undiluted test substance
- Evaluation (hr after challenge): 24 and 48 hours after challenge exposure

OTHER: None

Challenge controls:

10 animals treated with 100% Isopropyl alcohol

Positive control substance(s):

no

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100% Isopropyl alcohol

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no animals with positive responses

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100% Isopropyl alcohol. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no animals with positive responses.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100% Isopropyl alcohol

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no animals with positive responses

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100% Isopropyl alcohol. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no animals with positive responses.

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100% Isopropyl alcohol

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no animals with positive responses

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100% Isopropyl alcohol. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no animals with positive responses.

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

100% Isopropyl alcohol

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no animals with positive responses

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100% Isopropyl alcohol. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no animals with positive responses.

Interpretation of results:

not sensitising

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Substance did not induce sensitization in the guinea pig model.

Executive summary:

One group of 20 test animals was treated with undiluted Isopropyl alcohol for a period of 6 hours weekly for 3 induction exposures. The test animals and control animals were challenged with undiluted Isopropyl alcohol. No skin reactions were observed in the test and control animals therefore, it was concluded that Isopropyl alcohol is not a sensitizer.

Reference 5

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Propyl alcohol/Propan-1-ol is a very close analogue of isobutyl alcohol which is used in the manufacture of O-isobutyl ethylthiocarbamate (IBETC).Therefore, the health effects of propyl alcohol need to be considered in the assessment of O-isobutyl ethylthiocarbamate (IBETC) .

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

(no pretreatment of skin with SDS before topical induction, time of evaluation after challenge not given)

GLP compliance:

no

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

This test determined the potential of the test substance to be sensitizing to skin in the guinea pig model.

Species:

guinea pig

Strain:

Hartley

Sex:

not specified

Details on test animals and environmental conditions:

TEST ANIMALS
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- not reported

Route:

other: intradermal and epicutaneous occlusive

Vehicle:

no data

Concentration / amount:

INDUCTION (intradermal and topical): 100%
CHALLENGE: 100%

Route:

epicutaneous, occlusive

Vehicle:

no data

Concentration / amount:

INDUCTION (intradermal and topical): 100%
CHALLENGE: 100%

No. of animals per dose:

Test group: 15
control group: 6

Details on study design:

MAIN STUDY
A. INDUCTION EXPOSURE
INTRADERMAL INDUCTION
- No. of exposures: 2
- Test groups: a). substance undiluted, b).FCA, c). Test substance in FCA
- Control group: a). vehicle undiluted, b).FCA, c). vehicle in FCA (unclear what was used as vehicle since test substance was applied at a concentration of 100%)
- Site: no data
- Frequency of applications: no data
- Concentrations: 100%

TOPICAL INDUCTION (occlusive)
- Day(s) after intradermal induction: 7 days after
- No. of exposures: 1
- Test groups: undiluted test substance applied to skin
- Control group: undiluted vehicle applied to skin (unclear what was used as vehicle since test substance was applied at a concentration of 100%)
- Site: same site as intradermal induction
- Duration: 48h
- Concentrations: 100%

B. CHALLENGE EXPOSURE (topical occlusive)
- Day(s) after topical induction: 14 d
- Day of challenge: 21 days after intradermal induction
- No. of exposures: 1
- Exposure period: 24h
- Test groups: undiluted test substance applied to skin
- Control group: undiluted vehicle applied to skin (unclear what was used as vehicle since test substance was applied at a concentration of 100%)
- Site: naive skin
- washing of skin after application: no data
- Concentrations: 100%
- Evaluation (hr after challenge): no data

OTHER:
if neccessary a rechallenge was planned 7 day after challenge

Positive control substance(s):

yes

Remarks:

Dinitrochlorobenzene (intra dermal induction: 0.1%; topical induction; 0.1%; challenge; 0.1%), vehicle; propylene glycol

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

15

Clinical observations:

no data

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 15.0. Clinical observations: no data.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

6

Clinical observations:

no data

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 6.0. Clinical observations: no data.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.1% in propylene glycol

No. with + reactions:

15

Total no. in group:

15

Clinical observations:

no data

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.1% in propylene glycol. No with. + reactions: 15.0. Total no. in groups: 15.0. Clinical observations: no data.

Interpretation of results:

not sensitising

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The sustance did not induce sensitization in the guinea pig model.

Reference 6

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

(no pretreatment of skin with SDS before topical induction, time of evaluation after challenge not given)

GLP compliance:

no

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

This test determined the potential of the test substance to be sensitizing to skin in the guinea pig model.

Species:

guinea pig

Strain:

Hartley

Sex:

not specified

Details on test animals and environmental conditions:

TEST ANIMALS
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- not reported

Route:

other: intradermal and epicutaneous occlusive

Vehicle:

no data

Concentration / amount:

INDUCTION (intradermal and topical): 100%
CHALLENGE: 100%

Route:

epicutaneous, occlusive

Vehicle:

no data

Concentration / amount:

INDUCTION (intradermal and topical): 100%
CHALLENGE: 100%

No. of animals per dose:

Test group: 15
control group: 6

Details on study design:

MAIN STUDY
A. INDUCTION EXPOSURE
INTRADERMAL INDUCTION
- No. of exposures: 2
- Test groups: a). substance undiluted, b).FCA, c). Test substance in FCA
- Control group: a). vehicle undiluted, b).FCA, c). vehicle in FCA (unclear what was used as vehicle since test substance was applied at a concentration of 100%)
- Site: no data
- Frequency of applications: no data
- Concentrations: 100%

TOPICAL INDUCTION (occlusive)
- Day(s) after intradermal induction: 7 days after
- No. of exposures: 1
- Test groups: undiluted test substance applied to skin
- Control group: undiluted vehicle applied to skin (unclear what was used as vehicle since test substance was applied at a concentration of 100%)
- Site: same site as intradermal induction
- Duration: 48h
- Concentrations: 100%

B. CHALLENGE EXPOSURE (topical occlusive)
- Day(s) after topical induction: 14 d
- Day of challenge: 21 days after intradermal induction
- No. of exposures: 1
- Exposure period: 24h
- Test groups: undiluted test substance applied to skin
- Control group: undiluted vehicle applied to skin (unclear what was used as vehicle since test substance was applied at a concentration of 100%)
- Site: naive skin
- washing of skin after application: no data
- Concentrations: 100%
- Evaluation (hr after challenge): no data

OTHER:
if neccessary a rechallenge was planned 7 day after challenge

Positive control substance(s):

yes

Remarks:

Dinitrochlorobenzene (intra dermal induction: 0.1%; topical induction; 0.1%; challenge; 0.1%), vehicle; propylene glycol

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

15

Clinical observations:

no data

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 15.0. Clinical observations: no data.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

6

Clinical observations:

no data

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 6.0. Clinical observations: no data.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.1% in propylene glycol

No. with + reactions:

15

Total no. in group:

15

Clinical observations:

no data

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.1% in propylene glycol. No with. + reactions: 15.0. Total no. in groups: 15.0. Clinical observations: no data.

Interpretation of results:

not sensitising

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The sustance did not induce sensitization in the guinea pig model.

Reference 7

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS: Mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks (beginning of treatment)
- Weight at study initiation: 18.3 - 23.3 g
- Housing:single caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst / Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf), ad libitum
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg)
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): relative humidity 45-65%
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.

Vehicle:

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

Concentration:

The highest test item concentration which can be technically used was 100% of the undiluted test item. The dilutions were formulated in acetone/olive oil (4:1 v/v).
In the pre-test, two mice were treated with test item concentrations of 50 or 100%.
The test item in the main study was assayed at 25, 50, and 100%.

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration which can be technically used was 100% of the undiluted test item. The dilutions were formulated in acetone/olive oil (4:1 v/v).
In the pre-test, two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 50 or 100% once daily each on three consecutive days. At those concentrations the animals did not show signs of systemic toxicity or local irritation.

MAIN STUDY:

TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 25, 50, and 100% (w/v) in acetone:olive oil (4:1). The application volume, 25 µl, was spread over the entire dorsal surface (~ 8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
ADMINISTRATION OF ³H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED ³H-METHYL THYMIDINE

Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after treatment with ³HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a ß-scintillation counter.

INTERPRETATION OF RAW DATA

The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of ³HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
OBSERVATIONS

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
- Mortality / Viability: Once daily (week day) from experimental start to necropsy.
- Body weights: In the pre-test prior to the first application and prior to sacrifice; in the main experiment prior to the first application and prior to treatment with 3HTdR.
- Clinical signs (local / systemic): In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. In the main experiment clinical signs were recorded within 1 hour each application, and 24 ± 4 hours after the first and second application as well as on the day of preparation. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Positive control results:

Experiment performend in November 2009:
5, 10, and 25% alpha-hexyl cinnamic aldehyde in acetone:olibe oil (4:1) yielded a S.I. of 1.78, 2.54, and 4.88, respectively. The EC3 value calculated was 12.9%.
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

In this study Stimulation Indices of 1.22, 0.99, and 3.46 were determined with the test item at concentrations of 25, 50, and 100% in acetone/olive oil (4:1 v/v), respectively. A conventional dose response curve could not be established and an EC3 value was not calculated because of the odd dose response.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: As depicted in the table below.

Calculation and results of individual data; Vehicle: acetone/olive oil (4:1 v/v)

Test item concentration % (w/v)	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b )	S.I.
---	BG I	17	---	---	---	---
---	BG II	17	---	---	---	---
---	1	3517	3500	8	437.5	---
25	2	4282	4265	8	533.1	1.22
50	3	3479	3462	8	432.8	0.99
100	4	12129	12112	8	1514.0	3.46

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

 

Interpretation of results:

other: not sensitising

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Based on the numerical values of the stimulation indices one of which is slightly above 3, the test item Carbon disulfide has to be considered a skin sensitizer under the test condidions of this study. However, a conventional dose-response curve could not be established. An EC3 value was not calculated because of the odd dose-response.These factors contribute also in determining whether a borderline result shall be deemed as positive. In the opinion of the applicant this is a false positive and CS2 cannot be considered as a skin sensitizer base don this study.

Executive summary:

In this study carbon disulfide dissolved in acetone/olive oil (4:1 v/v) was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay according to OECD guideline 429 was performed using test item concentrations of 25, 50, and 100%. All treated animals survived the scheduled study period and no signs of systemic toxicity or local irritation were observed. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration results in a 3-fold or greater increase in incorporation of ³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices of 1.22, 0.99, and 3.46 were determined with the test item at concentrations of 25, 50, and 100% in acetone/olive oil (4:1). Based on the numerical values of these stimulation indices, the test item carbon disulfide has to be considered a skin sensitizer under the test conditions of this study. However, a conventional dose-response curve could not be established. An EC3 value was not calculated because of the odd dose-response. These factors contribute also in determining whether a borderline result shall be deemed as positive. In the opinion of the applicant this is a false positive and CS2 cannot be considered as a skin sensitizer base don this study.

Reference 8

Endpoint:

skin sensitisation, other

Remarks:

QSAR model

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1.SOFTWARE: OASIS v1.3

2. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: CAS No 55860-53-2, Smiles Code: O(C(=NCC)S)CC(C)C

3. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: skin sensitization
- Unambiguous algorithm: skin sensitization with autoxidation
- Defined domain of applicability: O-isobutyl ethylthiocarbamate is in the applicability domain of the model
- Appropriate measures of goodness-of-fit and robustness and predictivity: For substances in the applicability domain, a predictivity of 100 % was found for 100 industrial chemicals for the distinction of non-sensitizers of GHS Category 1.
- Mechanistic interpretation: Model predictions are based on simulation of skin metabolism and covalent interactions with proteins.

4. APPLICABILITY DOMAIN
- Descriptor domain: The chemical fulfills the general properties requirements and is in the interpolation structural space.
- Structural and mechanistic domains: N/A
- Similarity with analogues in the training set: not reported

5. ADEQUACY OF THE RESULT
The substance falls within the applicability domain of the model and the predictivity of the model for substance within its applicability domain was found to be reliable. Therefore, the preditction of O-isobutyl ethylthiocarbamate as a non-sensitizer is considered acceptable for the purposes of EC 1907/2006 and classification and labelling according to GHS criteria.

For further information see attached files for justification (QSAR)

Qualifier:

according to guideline

Guideline:

other: QSAR Toolbox Version 3.3.5.17

Principles of method if other than guideline:

A quantitative structure-activity relationship (QSAR) system for the estimation of the skin sensitization potency that incorporates skin metabolism and consideres the potential of parent chemicals and/or their activated metabolites to react with skin proteins. A chemically divers training set was used and their skin sensitization potency assigned to one of three classes.
The profiler was developed by National Institute of Technology and Evaluation (NITE) in the contract research project “Development of Hazard Assessment Techniques by using Structure-activity Method (FY2007-FY2011)” by New Energy and Industrial Technology Development

GLP compliance:

no

Remarks:

not applicable QSAR method for chemicals properties assessment.

Specific details on test material used for the study:

Substance description:
Common Name: IBETC
EC Number: 259-869-1
EC Name: O-isobutyl ethylthiocarbamate
CAS Number: 55860-53-2
IUPAC Name: O-isobutyl ethylthiocarbamate
Molecular formula:C7H15NOS
SMILES: O(C(=NCC)S)CC(C)C

Species:

mouse

Strain:

other: QSAR model

Sex:

male/female

Key result

Parameter:

EC3

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Cellular proliferation data / Observations:

This grouping method contains simple categories for skin sensitisation toxicity. This method is relevant for skin sensitisation toxicity endpoints in mammals.
The database include a set of 91 chemicals that have been evaluated for their skin sensitisation toxicity potential.

The QSAR program calculated a negative sensitization potential of the test substance.

OASIS v1.3 assessed O-isobutyl ethylthiocarbamate and one metabolite as non sensitiser.

Interpretation of results:

other: not sensitising

Conclusions:

Protein binding alerts for skin sensitization by OASIS v1.3-No alert found. Not sensitising
ADEQUACY OF THE RESULT
The substance falls within the applicability domain of the model and the predictivity of the model for substance within its applicability domain was found to be reliable. Therefore, the preditction of O-isobutyl ethylthiocarbamate as a non-sensitizer is considered acceptable for the purposes of EC 1907/2006 and classification and labelling according to GHS criteria.

Executive summary:

Profiling results:

DNA binding by OECD- No alert found

Est rogen Receptor Binding-Non binder, non cyclic structure

OECD HPV Chemical Categories-Not categorized

Protein binding for skin sensitization by OECD-No alert found

Protein binding potency-Not possible to classify according to these rules (GSH)

Superfragments-No superfragment

Toxic hazard classification by Cramer (original)-High (Class III)

US-EPA New Chemical Categories-Not categorized

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation

 

One group of 20 test animals was treated with undiluted substance for a period of 6 hours weekly for 3 induction exposures. The test animals and control animals were challenged with undiluted substance. No skin reactions were observed in the test and control animals therefore, it was concluded that the substance is not a sensitizer.

Substance did not induce sensitization in the guinea pig model.

Propan-2-ol (Isopropyl alcohol) is a very close analogue of isobutyl alcohol which is used in the manufacture of O-isobutyl ethylthiocarbamate (IBETC).Therefore, the health effects of isopropyl alcohol need to be considered in the assessment of O-isobutyl ethylthiocarbamate (IBETC).

 

Synopsis

Not sensitising

 

Respiratory sensitisation.

Due to the absence of chemical groups or other structural alerts this substance is not considered to exhibit an high hazard potential.O-isobutyl ethylthiocarbamate (IBETC)  is of low priority for further work based on a low hazard potential.is of low priority for further work based on a low hazard potential. Therefore testing for Respiratory sensitisation does not need to be performed.  

Respiratory sensitisation

Link to relevant study records

Reference

Endpoint:

respiratory sensitisation: in vivo

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Respiratory sensitisation.

Due to the absence of chemical groups or other structural alerts this substance is not considered to exhibit an high hazard potential.O-isobutyl ethylthiocarbamate (IBETC)  is of low priority for further work based on a low hazard potential.is of low priority for further work based on a low hazard potential. Therefore testing for Respiratory sensitisation does not need to be performed.  

Justification for classification or non-classification

Based on the hazard assessment of O-isobutyl ethylthiocarbamate (IBETC)  section 2.1 and 2.2. in IUCLID 6., available data for the substance and following the “Guidance on Information Requirement and Chemical Safety Assessment R.8. Characterisation of dose [concentration]- response for human health” andaccording to the criteria described in Directive 67/548 and in the CLP Regulation:

Directive 67/548

Respiratory Sensitisation Xn R42 May cause sensitization by inhalation Respiratory Irritation Xi R37 irritating to respiratory system

CLP	Respiratory Sensitisation H334 Resp. Sens. 1 May cause allergy or asthma symptoms or breath-ing difficulties if inhaled Respiratory Irritation H335 STOT SE 3 May cause respiratory irritation

It is concluded that the substance O-isobutyl ethylthiocarbamate (IBETC) does not meet the criteria to be classified for human health hazards for Inhalation - local effect: skin and respiratory sensitisation.