Chinomethionate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April 2018 to 27 April 2018 (Main LLNA experimental start and experimental completion)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Temp & relative humidity outside stated ranges, 19-25°C & 30-70%, respectively. Positive control group was part of a concurrent study where biopsy weights were taken, biopsy weights were therefore determined. No impact on study integrity or validity.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Temp & relative humidity outside stated ranges, 19-25°C & 30-70%, respectively. Positive control group was part of a concurrent study where biopsy weights were taken, biopsy weights were therefore determined. No impact on study integrity or validity.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Osaka Soda Co., Ltd. Japan
- Lot/batch No.of test material: 160722-01
- Expiration date of the lot/batch: 22 July 2018
- Purity test date: 3 August 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25 ºC, below 70% relative humidity)
- Stability under test conditions: Assumed stable for the duration of the study
- Solubility and stability of the test substance in the solvent/vehicle: Soluble in N,N-dimethylformamide (DMF) at 25% (w/v). Stability of the test item in the vehice not determined.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Solubilised at a maximum of 25% (w/v) in DMF. Formulations appeared to be a suspension by visual examination.

Species:

mouse

Strain:

CBA

Remarks:

CBA/CaOlaHsd mice

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Not specified. Only healthy animals were used for the study, the health status was certified by a veterinarian.
- Age at study initiation: 12 weeks old (age-matched, within one week) for the main LLNA study
- Weight at study initiation: 20.5 - 23.4 g (weight variation in study animals did not exceed ±20 % of the mean weight).
- Housing: Type II. polypropylene / polycarbonate. Bedding (Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Group caging, mice were provided with glass tunnel-tubes
- Diet: ad libitum .
- Water: ad libitum:
- Acclimation period: Acclimatization time: 35 days
- Indication of any skin lesions: None, only healthy animals were used and the health status was certified by a veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16.6 – 26.8°C
- Humidity (%): 26 - 69 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.
- IN-LIFE DATES: From: Main LLNA from 18 April 2018 to 27 April 2018 (Experimental start to experimental complation)

Vehicle:

dimethylformamide

Remarks:

DMF

Concentration:

The 25% (w/v) test item formulation was chosen as the highest suitable concentration for the test. Formulations appeared to be a solution by visual examination.
Three groups received Chinomethionate (formulated in DMF) at 25% (w/v), 10% (w/v) and 5 % (w/v) concentrations respectively.

No. of animals per dose:

In the main LLNA assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals.
- three groups received Chinomethionate (formulated in DMF) at 25% (w/v), 10% (w/v) and 5 % (w/v).
- one group, the negative control received the vehicle (DMF) only,
- one group, the positive control group received 25 % (w/v) HCA (dissolved in DMF)

Details on study design:

PRE-SCREEN (Prelim) TESTS:
- Compound solubility: Based on the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of OECD 429, the chosen vehicle was N,N-dimethylformamide (DMF). The 25% (w/v) test item formulation was chosen as the highest suitable concentration. Formulations appeared to be a solution by visual examination. In the Preliminary Irritation/Toxicity Tests performed with CBA/CaOlaHsd mice using two doses (2 animals/dose) at 25% and 10% (w/v) in DMF. The preliminary experiments were conducted in a similar experimental manner to the main study, but were terminated on Day 6 with no radioactive proliferation assay performed. The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429.

In the Preliminary Irritatiion all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.

- Irritation: No indications of any irritancy at the site of application. Test item precipitate was observed on the ears of all animals of the examined dose groups on Days 2-3 or in case of one animal of the 25% (w/v) dose group on Days 1-3.
- Systemic toxicity: No mortality or signs of systemic toxicity were observed.
- Ear thickness measurements: yes, ear thickness of animals was measured using a thickness gauge on Days 1, 3 and 6, and by ear punch weight determination after euthanasia of the experimental animals on Day 6. Slightly increase ear thickness values was observed for one animal of the 25%(w/v) dose group and for the right ear of the other animal of this dose group, however the revealing ear punch weight and the ear thickness increase were below the limit of positive (≥ 25 %).
The draining auricular lymph nodes of the animals were visually examined: they were larger than normal for both animals of the 25% (w/v) and for one animal of the 10% (w/v), they were slightly enlarged than normal for the other animal of the 10% (w/v) (subjective judgement by analogy with observations of former experiments).

Based on these results, 25% (w/v) dose is selected as top dose for the main test. Additionally, ear thickness of the experimental animals in the main test were measured by using a thickness gauge on Days 1 and 6.

- Erythema scores: No erythma was observed in all Preliminary Irritation/ Toxicity Test animals. Excessive local skin irritation is indicated by an erythema score ≥ 3 and/or an increase in ear thickness of ≥ 25 % on any day of measurement. All recorded values for for the increase in ear thickness and erythma in preliminary Irritation / Toxicity Test amimals were below criteria set values. In addition, no marked body weight loss was observed in the preliminary experiment.

MAIN STUDY

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:
- three groups received Chinomethionate (formulated in DMF) at 25% (w/v), 10% (w/v) and 5 % (w/v).
- the negative control group received the vehicle (DMF) only, the positive control group received 25 % (w/v) HCA (dissolved in DMF).

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA (OECD 429)
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of Citoxlab Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 μL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. The cell proliferation in the local lymph nodes was assessed by measuring disintegrations per minutes after incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI) in comparison with the control group.
No mortality or systemic clinical signs were observed during the main study. Test item precipitate or minimal amount of test item precipitate was observed on the ears of all animals of the 25%, 10% and 5% (w/v) dose groups on Days 1-3 or Day 1 and Day 3 or Days 2-3. There were no indications of any irritancy at the site of application. No treatment related effects were observed on the mean body weight changes in the main study. The ear thickness values were within the acceptable range for all the animals in the test item treated groups.

EVALUATION OF THE RESULTS
DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.

Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.The test item is regarded as a sensitizer if both of the following criteria are fulfilled:

- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- 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.
Since the test item gave a positive response and data permitted, the EC3 value of the test item was calculated (EC3 means the effective chemical concentration required for SI=3). The calculation of the EC3 value was conducted by log-linear interpolation according to the equation:

- Criteria used to consider a positive response:

The test item is regarded as a sensitizer if both of the following criteria are fulfilled:
- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- 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.
Since the test item gave a positive response and data permitted, the EC3 value of the test item was calculated (EC3 means the effective chemical concentration required for SI=3). The calculation of the EC3 value was conducted by log-linear interpolation:

Positive control substance(s):

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

Positive control results:

Test Group Name  Measured DPM/group  DPM Number of lymph nodes DPN Stimulation Index
 
Positive control
(25% (w/v) HCA 32684 32640.0 8 4080.0 8.2
 in DMF)

Negative control 4035 3991.0 8 498.9 1.0
(DMF)

The stimulation index values for the test item, Chinomethionate, were: 12.6, 11.9 and 10.0 at concentrations of 25%, 10% and 5% (w/v), respectively.

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Negative control (DMF)

Key result

Parameter:

SI

Value:

10

Test group / Remarks:

Chinomethionate: 5(w/v) % in DMF

Key result

Parameter:

SI

Value:

11.9

Test group / Remarks:

Chinomethionate: 10(w/v) % in DMF

Key result

Parameter:

SI

Value:

12.6

Test group / Remarks:

Chinomethionate: 25(w/v) % in DMF

Key result

Parameter:

EC3

Value:

0.4

Test group / Remarks:

The extrapolated EC3 value of Chinomethionate is 0.4% (w/v)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA

Test Group Name Measured DPM / group DPM Number of lymph nodes DPN Stimulation Index

Background
(5% (w/v) TCA) 49
39 - - - -
Negative control
(DMF) 4035 3991.0 8 498.9 1.0

Chinomethionate
5 (w/v) %
in DMF 39777 39733.0 8 4966.6 10.0
Chinomethionate
10 (w/v) %
in DMF 47572 47528.0 8 5941.0 11.9
Chinomethionate
25 (w/v) %
in DMF 50349 50305.0 8 6288.1 12.6

Positive control
(25% (w/v) HCA in DMF) 32684 32640.0 8 4080.0 8.2


DETAILS ON STIMULATION INDEX CALCULATION

DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = DPN value of a treated group divided by the DPN value of the negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

EC3 CALCULATION

The test item is regarded as a sensitizer if both of the following criteria are fulfilled:

- That exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- 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.
Since the test item gave a positive response and data permitted, the EC3 value of the test item was calculated (EC3 means the effective chemical concentration required for SI=3). The calculation of the EC3 value was conducted by log-linear interpolation according to the equation:

EC3ex = 2^{log2(c) + (3-d) x [log2(a) - log 2(c)]
(b-d)

where the lowest data point (lying immediately above the SI value of 3 on the LLNA dose-response plot) has the co-ordinates (c, d), and the next higher data point has the co-ordinates (a, b).

CLINICAL OBSERVATIONS:

No mortality or systemic clinical signs were observed during the main study. Test item precipitate or minimal amount of test item precipitate was observed on the ears of all animals of the 25%, 10% and 5% (w/v) dose groups on Days 1-3 or Day 1 and Day 3 or Days 2-3. There were no indications of any irritancy at the site of application.

BODY WEIGHTS

No treatment related effects were observed on the mean body weight changes in the main study. Marked body weight loss (≥5%) was observed for one animal of the 25% and 5% (w/v) dose group and for one animal of the positive control group. However, the mean body weight of these groups were in the acceptable range and these changes were considered as individual variability.


VALIDITY OF THE TEST
The result of the positive control substance α-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25 % in the relevant vehicle (DMF) using CBA/CaOlaHsd mice.
No mortality cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historical positive control data (stimulation index value of 8.2) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay. Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were in within the historical control range. Each treated and control group included 4 animals. The assay was considered valid.

Interpretation of results:

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

Conclusions:

In conclusion, under the conditions of the present assay, Chinomethionate, tested in a suitable vehicle, was shown to have a sensitisation potential (sensitizer) in the Local Lymph Node Assay. The extrapolated EC3 value of Chinomethionateis 0.4% (w/v).

The following classification/labelling is triggered:
Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 7) 2017: Category 1 (sub-category 1A).

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

It is not possible to conduct this study as the test substance is highly insoluble in water.

Justification for classification or non-classification

It is not possible to conduct this study as the test substance is highly insoluble in water.