Carbamodithioic acid, bis(mixed 2-ethylhexyl and 2-methylbutyl and pentyl) derivs., antimony (3+) salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 October 2020 thru 10 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Carbamodithioic acid, bis(mixed 2-ethylhexyl and 2-methylbutyl and pentyl) derivs., antimony (3+) salts

Molecular formula:

C33H66N3S6Sb to C51H102N3S6Sb

IUPAC Name:

Carbamodithioic acid, bis(mixed 2-ethylhexyl and 2-methylbutyl and pentyl) derivs., antimony (3+) salts

Test material form:

liquid: viscous

Specific details on test material used for the study:

PYSICAL DESCRIPTION: Highly viscous amber liquid.

PURITY: >99%

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: WT05-19
- Expiration date of the lot/batch: not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temp.
- Stability under storage conditions: stable for the duration of testing.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Received from Envigo RMS Inc. on October 14, 2020 (Preliminary Irritation Group Animals) and on October 14 and 21, 2020 (Test and Control Group Animals)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Preliminary Animals: Young adult (11 weeks); Test and Control Animals: Young adult (11-12 weeks)
- Weight at study initiation: 18.8 - 22.9 grams
- Housing: All caging conformed to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011)
- Diet (e.g. ad libitum): Envigo Teklad Global 16% Protein Rodent Diet® #2016. The diet was available ad libitum.
- Water (e.g. ad libitum): Filtered tap water was supplied ad libitum.
- Acclimation period: 14 or 21 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%):53 - 66
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12
- IN-LIFE DATES: From: To: 28 October 2020 thru 10 November 2020

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

10%, 25% and 50%

No. of animals per dose:

Preliminary Irritation (4 groups): 2 per group
Test (3 groups): 5 per group
Vehicle (Negative) Control: 5
Positive Control: 5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Solubility testing indicated that the test substance was soluble in Acetone/Olive Oil (4:1 v/v)
- Irritation: None at any dose
- Systemic toxicity: None at any dose
- Erythema scores: Draize method

MAIN STUDY

Criteria used to consider a positive response: The mean and standard deviation of the dpm values were calculated for each dose group. A stimulation index (SI) was derived for each experimental group by dividing the mean dpm of each experimental group by the mean dpm of the vehicle control group. Any test substance that produces an SI > 3 in the LLNA is normally considered “positive” for dermal sensitization potential (Kimber et al., 1994). All calculations were performed on the DPM minus background values. The EC3 value was not calculated since all dose levels induced a stimulation index of less than 3.0.


TREATMENT PREPARATION AND ADMINISTRATION:

Concentrations of 10%, 25% and 50% were selected for the main test based on results of the preliminary screening test. Dilutions of the test substance were prepared as w/w mixtures in Acetone/Olive Oil (4:1 v/v; AOO). The vehicle control, AOO, and a single concentration of a 25% w/w mixture of HCA in AOO were also prepared. All dosage preparations were freshly prepared on the day of application.

Positive control substance(s):

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

Statistics:

Statistical analysis was performed on the DPM minus background values. Significance was judged at p < 0.05. The treated groups and negative vehicle control group were compared using a One-Way Analysis of Variance (ANOVA), followed by comparison of the treated groups to control by Dunnett’s t-test for multiple comparisons. Where variances are considered significantly different by Bartlett’s test, groups were compared using a non-parametric method (Kruskal-Wallis non parametric analysis of variance followed by Dunn’s test) (INSTAT Biostatistics, Graph Pad Software, San Diego, CA). Outlier analysis was conducted using Grubbs (1969).

Results and discussion

Positive control results:

Very slight erythema (score of 1) was evident at four positive control sites on Day 3. Desquamation was present at two sites on Days 3 and 6.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

10% Concentration

Value:

0.74

Results 2

Key result

Parameter:

SI

Remarks:

25% Concentration

Value:

0.57

Results 3

Key result

Parameter:

SI

Remarks:

50% Concentration

Value:

0.96

Cellular proliferation data / Observations:

EC3 CALCULATION : Not calculated since all dose levels induced a stimulation index of less than 3.0

CLINICAL OBSERVATIONS: All animals were active and healthy throughout the study.

BODY WEIGHTS: Two mice each from the vehicle control and positive control groups and six from the test groups lost or failed to gain body weight during the study. All other mice gained body weight during the study.

DERMAL IRRITATION: For the high dose group only, very slight erythema (score of 1) was evident at one test site on Day 2 and at three sites on Day 3.

Any other information on results incl. tables

Group		Mean DPM minus background	SI	Sensitization Response
Vehicle Control	1	3034.91	-	N/A
Positive Control (25% HCA)	2	11737.68	3.87	Positive – valid study
10% Test Substance	3	2233.63	0.74	Not a Sensitizer
25% Test Substance	4	1727.84	0.57	Not a Sensitizer
50% Test Substance	5	2905.09	0.96	Not a Sensitizer

Group 5 - 50% Test Substance In AOO: Erythema/Edema Scores

Animal No.	Sex	Days
		1		2		3		6
		Left	Right	Left	Right	Left	Right	Left	Right
3621	F	0/0	0/0	0/0	0/0	1/0	1/0	0/0	0/0
3622	F	0/0	0/0	0/0	1/0	0/0	0/0	0/0	0/0
3623	F	0/0	0/0	0/0	0/0	0/0	0/0	0/0	0/0
3624	F	0/0	0/0	0/0	0/0	0/0	0/0	0/0	0/0
3625	F	0/0	0/0	0/0	0/0	0/0	1/0	0/0	0/0

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was not considered to be a contact dermal sensitizer at concentrations less than or equal to 50% in the mouse Local Lymph Node Assay (LLNA).

Executive summary:

In an in vivo Local Lymph Node Assay (LLNA) conducted in accordance with EU Method B.42 under GLP conditions with mice, test substance was not considered to be a contact dermal sensitizer at concentrations less than or equal to 50%. All animals were active and healthy throughout the study. Two mice each from the vehicle control and positive control groups and six from the test groups lost or failed to gain body weight during the study.  All other mice gained body weight during the study. For the high dose group only, very slight erythema (score of 1) was evident at one test site on Day 2 and at three sites on Day 3.