2,4-Dibromo-6-[(cyclohexyl-methyl-amino)-methyl]-phenylamine hydrobromide

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.12.2002 - 27.02.2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in GLP compliance and in accordance with an OECD guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,4-dibromo-6-{[cyclohexyl(methyl)amino]methyl}aniline hydrobromide

EC Number:

617-191-7

Cas Number:

81060-07-3

Molecular formula:

C14H20Br2N2 x HBr

IUPAC Name:

2,4-dibromo-6-{[cyclohexyl(methyl)amino]methyl}aniline hydrobromide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Bromhexine Hydrobromide
- Physical state: white powder
- Lot/batch No.: 02070864
- Expiration date of the lot/batch: Retest date: June 2003
- Storage condition of test material:
- Other: At room temperature, aerated rooms

Sampling and analysis

Analytical monitoring:

no

Test solutions

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
According to the results of a pre-experiment (without GLP) the test item was not soluble at a
concentration of 100 mg/L in test water and no homogeneous dispersion could be prepared.
Therefore, an undiluted filtrate of a supersaturated dispersion with the maximum
concentration of dissolved test item was used as the only test concentration. Thus, a Iimit
test was performed in accordance with the EU Commission Directive 92/69/EEC to
demonstrate that the test item has no toxic effect on the test organisms up to the highest
test item concentration which could be dissolved in test water. Additionally, a control was
tested in parallel (test water without test item).
The test medium was prepared as follows: A supersaturated dispersion with a loading rate of
100 mg/L was prepared by weighing 30.2 mg of the test item into 300 ml test water. No
auxiliary solvent or emulsifier was used. The test item was mixed into the test water as
homogeneously as possible by ultrasonic treatment for 15 minutes and by intense stirring for
48 hours at room temperature in the dark to dissolve a maximum concentration of the test
item in the dispersion (according to the "Guidance Document on Aquatic Toxicity Testing of
Difficult Substances and Mixtures"; OECD Series on Testing and Assessment No. 23
(2000)).
Then, the supersaturated stock dispersion of the test item was fi ltered through a membrane
filter (Schleicher & Schuell, Type NC45, pore size 0.45 ~m) under vacuum. The test medium
was prepared just before the introduction of the daphnids (=start of the test). On request of
the sponsor the concentrations of the test item were not analytically verified during the test
period

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus
- Strain: clone 5
- Source: originally supplied by the University of Sheffield/UK in 1992, Since this date the clone is bred in the Iaberateries of RCC
- Age at study initiation (mean and range, SD): 6- 24 hours
- Feeding during test: None

Study design

Test type:

static

Limit test:

yes

Total exposure duration:

48 h

Test conditions

Hardness:

2.5 mmoi/L (= 250.0 mg/L) as CaC03

Test temperature:

20 C

pH:

7.8 - 7.9

Dissolved oxygen:

8.5 - 8.7

Salinity:

CaCl2 x 2H20: 2.0 mmoi/L (= 294.0 mg/L)
MgS04 X 7H20: 0.5 mmoi/L (= 123.0 mg/L)
NaHC03: 0.75 mmoi/L (= 65.0 mg/L)
KCI: 0.075 mmoi/L (= 5.8 mg/L)
Alkalinity: 0.8 mmoi/L

Nominal and measured concentrations:

nominal: 100mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 ml glass beakers
- Type (delete if not applicable): open / closed: covered with glass plates
- fill volume: 50 ml
- Aeration: The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period the test water was not aerated.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: lower than one daphnia per 2 ml test solution


OTHER TEST CONDITIONS
A 16-hour light to 8-hour darkness photoperiod (with a 30 minute
transition period). Light intensity in the light period between 550 and
685 Lux.


TEST CONCENTRATIONS
100 mg/ml ( filtrate of supersaturated dispersion)

Reference substance (positive control):

yes

Remarks:

potassium dichromate is tested as a positive control at least once a year

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Any other information on results incl. tables

The daphnids were observed for immobility after 24 and 48 hours of exposure (those organisms not able to swim within 15 seconds after gentle agitation of the test beaker are considered tobe immobile).

The EC50 and EC100 could not be calculated due to the absence of toxicity of the test item.

The NOEC. ECO were determined directly from the raw data.

In the control and in the undiluted filtrate of the test item (with a loading rate of 100 mg/L) no

immobilized test organisms or other signs of intoxication were determined during the test

period of 48 hours

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

ln conclusion , the test item Bromhexine Hydrobromide had no acute toxic effects on Daphnia magna up to its solubility Iimit in test water under the present conditions.
No remarkable observations were made concerning the appearance of the test medium. The testmedium was a clear solution throughout the whole test duration