5-bromo-1,3-dichloro-2-fluorobenzene

Endpoint summary

Description of key information

Sensitising to the skin (in vivo), Cat. 1B (GLP, OECD TG 429)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

The study was conducted to meet the national regulatory requirements in a non-EEA country.

Type of information:

experimental study

Adequacy of study:

key study

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)

Version / remarks:

2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Age/weight at dosing: Young adult, Test and Control Animals (9-10 weeks)/ 17.4-22.9 grams at experimental start (females)
Source: Envigo RMS Inc.
Housing: Individually housed in plastic solid bottom cages during the dosing and resting phase of the study. After final weighing until sacrifice, animals were housed in their respective dose groups in plastic cages with bedding
Acclimatisation period: 7 - 21 days
Diet: Envigo Teklad Global 16% Protein Rodent Diet® #2016. The diet was available ad libitum.
Water: Filtered tap water ad libitum
Environmental conditions: Temperature: 19-23 °C
Humidity: 56-60%
Air changes: 13/hour
Photoperiod: 12 hours light / 12 hours dark
In-life dates: Start: May 16, 2018; End: July 26, 2018

Vehicle:

other: 1% Pluronic® L92

Concentration:

Concentrations of 5%, 10% and 25% 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 1% Pluronic® L92

No. of animals per dose:

4

Details on study design:

Preliminary Toxicity Testing
Three mice were treated with the test substance at the maximum concentration suitable for application (100%) and at 50% and 25% dilutions. The ears of the mice were evaluated for erythema and edema immediately prior to dosing on Days 1, 2, 3, and/or on Day 6 according to the scoring system described in Table 16. Body weight measurements were taken on Days 1 and/or 6 (see also Deviation ). Ear thickness measurements were taken on Day 1
(pre-dose), Day 3 and/or Day 6.
Twenty-five L of the test substance was applied to the dorsum of both ears of the mice once per day for three consecutive days. Application was done using an appropriate size micropipette to accurately deliver 25 L. The dose was gently spread as evenly as possible over the dorsal surface of the ear using the disposable pipette tip. No treatment was made on Days 4 and 5. On Day 6, each site was evaluated for local reactions (erythema & edema).
The animal was observed daily for signs of toxicity. The Study Director used this data in conjunction with any pre-existing data to select the three concentrations to be tested. The test substance at 10%, 25%, and 50% (w/w) mixtures in 1% Pluronic® L92 were selected for test.
Selection of Animals/Dose Levels
Prior to dosing, the animals were weighed and the ears were checked for any abnormalities or clinical signs of diseases or injury. Sixteen healthy, naive female mice without pre-existing ear irritation were selected and distributed (four mice per group) into the following groups:
Group 1: vehicle control, 0% w/w
Group 2: test substance, 5% w/w
Group 3: test substance, 10% w/w
Group 4: test substance, 25% w/w
Concentrations were selected based on toxicity, solubility, irritancy, and viscosity.
Sample Preparation
Concentrations of 5%, 10% and 25% 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 1% Pluronic® L92. The vehicle control, 1% Pluronic® L92 was also prepared. All dosage preparations were freshly prepared on the day of application.
Test Substance Application
Beginning on Day 1, a quantity of 25 µL of the appropriate test substance concentration or the vehicle alone was applied to the dorsum of both ears of each mouse once per day for three consecutive days (Days 1, 2, and 3) using a micropipette. During application, the material was gently spread as evenly as possible over the dorsal surface of the ear using the micro-pipette tip.
Dermal Scoring
Prior to each application (Days 1, 2, and 3) and on Day 6, the ears were evaluated for erythema and edema according to the modified Draize scoring system (Draize, Woodard, & Calvary, 1944)
Ear Thickness Measurements
Duplicate measurements of each animal’s ears were made using a micrometer. The measurements were made at the apex of the pinna. Measurements were taken on the preliminary screen animals on Day 1 (pre-dose), Day 3 and/or Day 6. The % ear swelling was calculated for each ear using the following equation:
% Ear swelling =(B-A)/A x 100%, where
A = ear thickness measurement on Day 1 (mm x 0.01)
B = ear thickness measurement on Day 3 or 6 (mm x 0.01)

3H-methyl Thymidine Injections
On Day 6 of the study (three days after the final topical application) 250 µL of sterile phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine was injected intravenously via the tail vein of each mouse.
Lymph node assessment
Approximately five hours after the injection, all test and control mice were euthanized via overdose of inhaled Isoflurane and the draining auricular lymph nodes from all animals were excised. The lymph nodes were evaluated for each individual mouse. A single cell suspension of lymph node cells (LNC) was prepared in PBS by gently massaging the lymph nodes between the frosted ends of two microscope slides over a collection vessel. The slides were then rinsed briefly with PBS into the vessel. The contents of the vessel were transferred to a centrifuge tube and washed with an excess of PBS and centrifuged for approximately 10 minutes at 1800 rpm, with an RCF of 489G. This process was carried out twice. In both cases, the supernatant was decanted and discarded following each centrifugation. After the second wash, 5 mL of the 5% trichloroacetic acid (TCA) in distilled water was then added to the sediment and the tube was vortexed briefly. The DNA was then precipitated in the 5% TCA in distilled water at approximately 4 °C overnight (approximately 18 hours).
Following the overnight precipitation of the DNA, the tubes were centrifuged again for approximately 10 minutes at 1800 rpm and the supernatant was discarded. The resulting precipitate was re-suspended using 1 mL of the 5% TCA in distilled water and transferred to 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine was measured by β-scintillation counting and expressed as disintegrations per minute, minus background dpm.
Clinical Observations
All test, control and preliminary mice were observed for signs of mortality, gross toxicity, and/or behavioral changes daily. Preliminary mice were euthanized via CO2 inhalation and all test and control mice were euthanized via overdose of inhaled Isoflurane (an anesthetic) on Day 6.
Body Weights
Individual body weights of the preliminary animals were recorded on Day 1 (initial) shortly before test substance application and prior to sacrifice on Day 6 . Individual body weights of test and control animals were recorded on Day 1 (initial) shortly before test substance application and prior to IV injections of 3H-methyl thymidine on test Day 6.

Positive control substance(s):

other: alpha-hexylcinnamaldehyde

Positive control results:

The positive control (HCA) at 25% produced a dermal sensitization response in mice (SI = 4.04).

Key result

Parameter:

SI

Value:

2.25

Test group / Remarks:

Group 2, 5% test substance in 1% Pluronic L92

Key result

Parameter:

SI

Value:

2.41

Test group / Remarks:

Group 3, 10% test substance in 1% Pluronic L92

Key result

Parameter:

SI

Value:

3.74

Test group / Remarks:

Group 4, 25% test substance in 1% Pluronic L92

Key result

Parameter:

EC3

Value:

16.7

Test group / Remarks:

Group 4

One preliminary mouse was euthanized for humane reasons due to systemic toxicity on Day 2.  Prior to death, the animal was hypoactive and exhibited hunched posture.  Gross necropsy of the deceased animal revealed no gross abnormalities.

All animals appeared active and healthy throughout the study. One mouse from the vehicle control and six mice in the test substance groups lost or failed to gain body weight during the study.  All other mice gained body weight during the study

Group 1 (Vehicle Control – 1% Pluronic® L92): No dermal irritation was observed for any of the vehicle control group sites.

Group 2 (5% Test Substance in 1% Pluronic® L92): No dermal irritation was observed for any of the test group sites.

Group 3 (10% Test Substance in 1% Pluronic® L92): No dermal irritation was observed for any of the test group sites.

Group 4 (25% Test Substance in 1% Pluronic® L92): Very slight erythema (score of 1) was evident at one test group site on Day 3.

Treatment of mice with 5%, 10% and 25% of CA5528 resulted in stimulation index values of 2.25, 2.41 and 3.74, respectively. As a stimulation index (SI) of greater than 3.0 was observed in the 25% treatment group, the test substance was considered positive for a dermal sensitization potential. The EC3 value was calculated to be 16.7%.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on these findings and on the evaluation system used, CA5528 is considered to be a contact dermal sensitizer in the LLNA. The EC3 value calculated for the test substance was 16.7%.

Executive summary:

The purpose of this skin sensitization study was to assess the possible allergenic potential of the substance when administered topically to the albino mouse, CBA/J.

Three concentrations (5%, 10% and 25%) of the test substance w/w in 1% Pluronic® L92 Surfactant w/w in distilled water (1% Pluronic® L92) and the vehicle alone were topically applied to sixteen healthy test mice (four mice/group) for three consecutive days. Three days after the last application, 250 µL of sterile phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine was injected intravenously via the tail vein of each mouse.  Approximately five hours later, all animals were euthanized via an overdose of inhaled Isoflurane and the draining (auricular) lymph nodes were harvested and prepared for analysis in a scintillation counter.  The results are presented in disintegrations per minute per mouse (dpm/mouse).  The ears of each animal were also evaluated for erythema and edema prior to each application and again on Day 6, prior to the 3H-methyl thymidine IV injection.

The sensitivity of the procedure was validated using recent historical positive control data (Study 47597).  A positive control group (four animals) was maintained under the same environmental conditions and treated in the same manner as the test and vehicle control animals.  The positive control group animals were treated with a 25% (w/w) mixture of alpha-hexylcinnamaldehyde (HCA), purity ≥ 95%, in 1% Pluronic® L92.  

There were no treatment related deaths and no signs of systemic toxicity observed in the animals.

The stimulation index was:

Group 1 - vehicle control: not applicable

Group 2 - 5% test substance: SI = 2.25

Group 3 - 10% test substance: SI = 2.41

Group 4 - 25% test substance: SI = 3.74

Based on the results of this study, CA5528 is considered to be a contact dermal sensitizer in the LLNA. The EC3 value calculated for the test substance was 16.7%. Proper conduct of the LLNA was confirmed via a positive response with 25% alpha-Hexylcinnamaldehyde, purity ≥ 95% (HCA), a moderate contact sensitizer.

The positive control (HCA) at 25% produced a dermal sensitization response in mice (SI=4.04).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance was positively tested in vivo in a local lymph node assay in mice and meets the criteria for classification for skin sensitisation Cat. 1B in accordance with CLP-Regulation (EC) No. 1272/2008.