Bromobenzene

Endpoint summary

Description of key information

WoE: not sensitising

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:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

limited data on results (body weights, local irritation, individual data/readouts)

Justification for type of information:

Please refer to analogue justification provided in IUCLID section 13

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 July 2010

Deviations:

yes

Remarks:

limited data on results (body weights, local irritation, individual data/readouts)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Remarks:

(CBA/CaOlaHsd and CBA/J, not further specified)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Haraln Winkelmann GmbH, Borchen, Germany or Charles River Laboratories, Sulzfeld, Germany GmbH (not further specified), Sulzfeld, Germany
- Females nulliparous and non-pregnant: Not specified
- Age at study initiation: 6-12 weeks
- Housing: Makrolon type I or II cages
- Diet: Provimi Kliba, (Kliba-Labordiät, Maus/Ratte Haltung 'GLP', SA, Kaiseraugst, Basel, Switzerland), ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

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

Concentration:

25, 50 and 100% (undiluted) (no further information available)

No. of animals per dose:

6

Details on study design:

PRE-SCREEN TESTS:
- Ear thickness measurements: conducted in three mice exposed to a concentration of 5% in order to determine skin irritating concentrations. The resultant high concentration and two lower concentrations spaced by a factor of approximately 3 each were selected for the study (if an indication of ear skin irritation was observed, lower concentrations were used). The concentrations tested in the presented study were higher than that defined in the published harmonized minimum performance standards (PS study).

Positive control substance(s):

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

eugenol (CAS No 97-53-0)

mercaptobenzothiazole (CAS No 149-30-4)

other: All substances defined as recommended reference substances for the LLNA performance standards defined in OECD TG 429 were tested in the conducted study.

Statistics:

The estimated concentration (EC) for the SI = 1.5 (defined for the non-radioactive evaluation method) or SI = 3 (defined for the standard radioactive LLNA procedure) was calculated by linear regression using the data points directly below and above the threshold or using the two nearest points below or above the SI. Results exceeding these cut-off SIs are considered to indicate biologically relevant signs of lymph node cell proliferation. A substance was considered a sensitizer if at least one concentration tested caused a concentration-dependent statistically significant and/or biologically relevant increase in LNCC, dpm and/or lymph node weight compared with the vehicle control.

Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The SIs of lymph node cell cound (LNCC, referred to as "non-radioactive" method, 3H-thymidine incorporation, lymph node weight (LNW) and ear weight were calculated as the ration of the test group mean values divided by those of the vehicle control group. The presence of a skin irritation reaction to topical treatment was determined by whether a greater than 20% increase in ear weight occurred.

Key result

Parameter:

SI

Remarks:

standard radioactive LLNA

Value:

>= 1.91 - <= 5.3

Test group / Remarks:

25, 50 and 100%

Remarks on result:

other: At the highest concentration tested, increased ear weight was determined. Thus, a positive response due to irritation cannot be excluded.

Parameter:

SI

Remarks:

non-radioactive LLNA

Value:

>= 0.97 - <= 1.61

Test group / Remarks:

25, 50 and 100%

Remarks on result:

other: At the highest concentration tested, increased ear weight was determined. Thus, a positive response due to irritation cannot be excluded.

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA:
EC1.5 (non-radioactive method): 79.0

EC3 CALCULATION
EC3 (radioactive method) = 45.6

Interpretation of results:

Chlorobenzene induced a dose-dependent increase in cell count (non-radioactive LLNA) and thymidine incorporation (standard LLNA). Test concentrations of 25, 50 and 100% resulted in SI values of 0.97, 1.34 and 1.61 for cell counts (non-radioactive LLNA) and SI values of 1.91, 3.23 and 5.30 for thymidine incorporation, respectively. In parallel, ear weights were increased by more than 20% at the highest test concentration, which indicates skin irritation. Considering that chlorobenzene is classified as skin irritant (category 2), a false-positive result due to skin irritation cannot be excluded for both evaluation methods. Taking further into account that 1) chlorobenzene revealed negative results in the studies considered for the recommended reference substance list cited in OECD TG 429 and 2) the dose tested are higher than that defined in the published harmonized minimum performance standards, the obtained result is considered as false-positive result. Therefore, chlorobenzene is presumed to be a non-sensitizer in the conducted LLNA test. 

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

CLP: Not classified

Reference 2

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

Justification for type of information:

Please refer to the analogue approach justification document provided in section 13

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: The local lymph node assay using 5-bromo-2-deoxyuridine (BrdU) with flow cytometry (LLNA: BrdU-FCM) is a modified LLNA that is used to identify skin sensitizers by counting BrdU-incorporated lymph node cells with flow cytometry.
Proliferation of lymph node cells after topical application of a test substance is measured by comparing the mean proliferation in each test group to the mean proliferation in the vehicle treated control group. The ratio of the mean proliferation in each treated group to that in the concurrent vehicle control group, termed the SI, is determined. The method is based on the use of measuring BrdU content to indicate an increased number of proliferating cells in the draining auricular lymph nodes. BrdU is an analogue of thymidine and is similarly incorporated into the DNA of proliferating cells. The incorporation of BrdU is measured by flow cytometer, which utilises an antibody specific for BrdU that is also labelled with the fluorochrome fluorescein isothiocyanate (FITC), that can be measured using flow cytometer.
- Short description of test conditions: The test item was applied to the entire dorsal surface of each ear of each mouse for 3 consecutive days. On day 5 mice were intraperitoneally injected with BrdU solution in PBS and sacrificed after 24 ± 2 h. Auricular lymph nodes were collected from which the lymph node cells were isolated. Incorporated BrdU was analyzed using a flow cytometry.
- Parameters analysed / observed: ear thickness, BrdU incorporation

GLP compliance:

no

Remarks:

not specified in publication

Type of study:

other: LLNA: BrdU-FCM

Species:

mouse

Strain:

CBA:J

Remarks:

(BALB/c strain was additionally tested)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Koatech, Pyeongtaek, Korea (CBA:J), Samtako, Osan, Korea (BALB/c)
- Microbiological status of animals: specific pathogen free
- Age at study initiation: 7 weeks
- Housing: in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care International Animal Care Policies
- Diet: solid diet (Purina Mills Inc. Seoul, Korea), ad libitum
- Water: sterilized water, ad libitum
- Acclimation period: 5 or 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1
- Humidity (%): 50 ± 10
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

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

Concentration:

CBA/J: 25, 50 and 100% (v/v)
BALB/c: 10, 25 and 50% (v/v)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: dissolved test item with maximum solubility was verified no suspension, layer or precipitation when it was mixed with the vehicle and leaved 5 minutes
- Irritation: an erythema score of over 2, a decrease in body weight by more than 5%, an increase in ear weight or thickness by more than 25% on day 6 compared with day 1 or death during the study was considered as severe skin irritation
- Systemic toxicity: same criteria as for severe skin irritation
- Ear thickness measurements: ear thickness was measured at the center of both ears using a thickness gauge on days 1, 3 and 6

MAIN STUDY
Results of the preliminary test:
1) Systemic toxicity or severe irritation: 10, 5, 2.5, 1 and 0.5% and below
2) Non-irritation: 100 and 50%

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 5-bromo-2-deoxyuridine incorporation determined by flow cytometer with an antibody based method
- Criteria used to consider a positive response: If the stimulation index (SI) was 2.7 or above (SI ≥ 2.7), a chemical was classified as a sensitizer. Chemicals with a stimulation index (SI) below 2.7 (SI<2.7) were classified as non-sensitizers

TREATMENT PREPARATION AND ADMINISTRATION:
25 µl of the test item was applied to the entire dorsal surface of each ear of each mouse. The application was repeated on days 2 and 3. On day 5 mice were intraperitoneally injected with 100 µl of a freshly prepared BrdU solution (20 mg/mL) in PBS and sacrificed after 24 ± 2 h. The central parts of both ears using a 6 mm biopsy punch were collected and weighed. Auricular lymph nodes were collected, weighed and put into a cell strainer on a 6-well plate filled with cold PBS. The auricular lymph nodes were mashed with a spatula to prepare lymph node cells.
Incorporated BrdU was analyzed using a FITC BrdU flow kit following the manufacturer’s instructions (BD Biosciences, San Jose, CA, USA). Viable lymph node cells were gated and a total of 10,000 gated cells were analyzed using BD FACS CaliburTM flow cytometry.

Positive control substance(s):

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

eugenol (CAS No 97-53-0)

mercaptobenzothiazole (CAS No 149-30-4)

other: All substances defined as recommended reference substances for the LLNA performance standards defined in OECD TG 429 were tested in the conducted study.

Statistics:

Statistical analyses of the changes in the number of lymph node cells and the proportion of BrdU-incorporated lymph node cells were performed using Graphpad Prism software (San Diego, CA, USA). A value of p<0.05 was considered statistically significant. The EC 2.7 or 3 described an estimated concentration needed to produce a stimulation index of 2.7 or 3.

Positive control results:

In CBA/J and BALB/c mice, 11/13 known skin sensitising substances (according to the reference list recommended in OECD TG 429) revealed a SI value >/gleich 2.7 and thus meet the reliability criteria. 2-mercaptobenzothiazole and methyl methacrylate failed the evaluation criteria for a positive response in both species.

Key result

Parameter:

SI

Value:

1.3

Variability:

0.53

Test group / Remarks:

25% (v/v)

Remarks on result:

other: CBA/J mice

Key result

Parameter:

SI

Value:

1.49

Variability:

0.36

Test group / Remarks:

50% (v/v)

Remarks on result:

other: CBA/J mice

Key result

Parameter:

SI

Value:

2.14

Variability:

0.67

Test group / Remarks:

100% (v/v)

Remarks on result:

other: CBA/J mice

Parameter:

SI

Value:

1

Variability:

0.45

Test group / Remarks:

Vehicle control

Remarks on result:

other: CBA/J mice

Key result

Parameter:

SI

Value:

1.12

Variability:

0.49

Test group / Remarks:

10% (v/v)

Remarks on result:

other: BALB/c mice

Key result

Parameter:

SI

Value:

1.25

Variability:

0.36

Test group / Remarks:

25% (v/v)

Remarks on result:

other: BALB/c mice

Key result

Parameter:

SI

Value:

1.67

Variability:

0.23

Test group / Remarks:

50% (v/v)

Remarks on result:

other: BALB/c mice

Parameter:

SI

Value:

1

Variability:

0.39

Test group / Remarks:

Vehicle control

Remarks on result:

other: BALB/c mice

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
No statistically difference was between the dose groups and the vehicle control was observed

DETAILS ON STIMULATION INDEX CALCULATION
SI = (BrdU incorporation rates of the test substance group x the number of lymph node cells (cells/mL)) / (BrdU incorporation rates of the negative control group x the number of lymph node cells (cells/mL))

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

CLP: not classified

Reference 3

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:

guideline study without detailed documentation

Remarks:

(no details on study conduct given, no rationale for dose selection given)

Justification for type of information:

Please refer to the analogue approach justification document provided in section 13

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 July 2010

Deviations:

yes

Remarks:

(no details on study conduct given, no rationale for dose selection given)

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

not specified

Sex:

not specified

Vehicle:

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

Concentration:

5, 10 and 25%

Positive control substance(s):

other: not specified, 106 test chemicals were tested with 73 of them being positive

Positive control results:

In total, 106 test chemicals were tested with 73 of them being positive. Thus, the applied test cond
itions are considered as valide.

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

5%

Remarks on result:

other: not sensitising

Key result

Parameter:

SI

Value:

1.7

Test group / Remarks:

10%

Remarks on result:

other: not sensitising

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

25%

Remarks on result:

other: not sensitising

Cellular proliferation data / Observations:

Topical application of 5, 10 and 25% chlorobenzene led to SI values of 1.1, 1.7 and 1.6, respectively.
Thus, chlorobenzene did not exhibit skin sensitising properties in the conducetd LLNA.

In this publication local lymph node data for 106 chemicals (among them chlorobenzene) are listed. Stimulation indices after exposure to 5, 10 and 25% chlorobenzene were 1.1, 1.7, and 1.6, respectively. Thus, based on the results of the conducted study, chlorobenzene was reported to be inactive in the lymph node assay (LLNA) and therefore not sensitising.

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Sensitisation

Justification for read-across

There are no data available regarding skin sensitisation of bromobenzene (CAS 108-86-1). Thus, read-across from an appropriate substance (chlorobenzene, CAS 108-90-7) is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII, 8.3.

Structural similarities and comparable toxicokinetic properties of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Skin sensitisation

CAS 108-90-7

The available data on the skin sensitising properties of chlorobenzene (LLNA, guinea pig test data and clinical evidence) reveal that the substance is not a skin sensitiser. Based on this data base, chlorobenzene was chosen as one of five negative reference substances for the conduct of performance standards according to OECD TG 429.

The skin sensitising properties of chlorobenzene (CAS 108-90-7) were tested in a study performed equivalent or similar to OECD TG 429 under GLP conditions using the murine local nymph node assay (LLNA, Basketter et al., 2012). In this publication local lymph node data for 22 chemicals (all substances are defined as recommended reference substances in OECD TG 429, among them chlorobenzene) were obtained. Groups of six mice were exposed daily, for three consecutive days, to 25, 50 and 100% chlorobenzene or to the vehicle alone, on the dorsum of both ears. Subsequently, mice were injected intravenously with [3H]-thymidine. Radioactivity was measured as a function of isotope incorporation in draining auricular lymph nodes. Additionally, lymph node cells counts (LNCC) were run in parallel (referred to as “non-radioactive method”). Chlorobenzene induced a dose-dependent increase in cell count (non-radioactive LLNA) and thymidine incorporation (standard LLNA). Test concentrations of 25, 50 and 100% resulted in SI values of 0.97, 1.34 and 1.61 for cell counts (non-radioactive LLNA) and SI values of 1.91, 3.23 and 5.30 for thymidine incorporation, respectively. In parallel, ear weights were increased by more than 20% at the highest test concentration, which indicates skin irritation. Considering that chlorobenzene is classified as skin irritant (category 2), a false-positive result due to skin irritation cannot be excluded for both evaluation methods. Taking further into account that 1) chlorobenzene revealed negative results in the studies considered for the recommended reference substance list cited in OECD 429 and 2) the doses tested are higher than that defined in the published harmonized minimum performance standards, the obtained result is considered as false-positive result. Therefore, chlorobenzene is presumed to be a non-sensitizer in the conducted LLNA test.

 

In further publication, the skin sensitising potential of chlorobenzene (CAS 108-90-7) was tested in a study using a modified murine local nymph node assay that is used to identify skin sensitizers by counting 5-bromo-2-deoxyuridine (BrdU)-incorporated lymph node cells by flow cytometry (LLNA: BrdU-FCM, Lee et al., 2017). This method is based on measuring BrdU content to indicate an increased number of proliferating cells in the draining auricular lymph nodes. In this publication, data for 18 chemicals ((all substances are defined as recommended reference substances in OECD TG 429, among them chlorobenzene)) were obtained. Groups of five mice were exposed daily, for three consecutive days, to 25, 50 and 100% of chlorobenzene or to the vehicle alone, on the dorsum of both ears. Subsequently, mice were intraperitoneally injected with BrdU solution. Auricular lymph nodes were collected and incorporated BrdU in the lymph node cells was measured using flow cytometry. BrdU stimulation indices after exposure to 25, 50 and 100% chlorobenzene were 1.30, 1.49 and 2.14, respectively. Thus, under the conditions of the test, the test substance chlorobenzene revealed no skin sensitising properties. In CBA/J and BALB/c mice, 11/13 known skin sensitising substances (according to the reference list recommended in OECD TG 429) revealed a SI value ≥ 2.7 and thus meet the reliability criteria. 2-mercaptobenzothiazole and methyl methacrylate failed the evaluation criteria for a positive response in both species.

In addition, the skin sensitisation potential of chlorobenzene (CAS 108-90-7) was tested in a further LLNA study performed according to OECD TG 429 (Ashby et al., 1995). In this publication local lymph node data for 106 chemicals (among them chlorobenzene) are listed. Mice were exposed daily for 3 consecutive days, to 5, 10 and 25% chlorobenzene or to the vehicle alone, on the dorsum of both ears. Subsequently, mice were injected intravenously with [3H]-thymidine and activity measured as a function of isotope incorporation in draining auricular lymph nodes. Stimulation indices after exposure to 5, 10 and 25% chlorobenzene were 1.1, 1.7, and 1.6, respectively. Thus, based on the results of the conducted study, chlorobenzene was reported to be inactive in the lymph node assay (LLNA) and therefore not sensitising.

 

 

Overall conclusion on skin sensitisation

The available experimental data did not indicate a sensitising potential of the structural analogue substance chlorobenzene. Thus, based on the analogue approach, bromobenzene is not considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the analogue approach, the available data on skin sensitisation do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.