Reaction products of m-phenylenebis(methylamine) with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 March 2018 - 17 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

23 March 2006; Annex 5 corrected 28 July 2011.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3- epoxypropane, reaction products with mphenylenebis(methylamine)

Cas Number:

113930-69-1

IUPAC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3- epoxypropane, reaction products with mphenylenebis(methylamine)

Test material form:

solid

Details on test material:

- Physical appearance: light amber solid
- Storage of test material: at room temperature

Specific details on test material used for the study:

- No correction factor for purity was required and therefore not applied.
- The test material is irritant/corrosive.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For determination of test concentrations extra vessels from the stock solutions, all test concentrations and the controls were incubated without organisms. From these vessels, single samples for possible analysis were taken according to the schedule below:
Frequency: at t=0 h, t=48 h and t=72 h
Volume: 20 mL from the approximate centre of the test vessels
Storage: Stock solutions were analysed on the day of sampling while test samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the test facility.

Test solutions

Vehicle:

yes

Remarks:

dimethyl sulfoxide

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Range-finding and final study: Stock solutions were prepared in dimethyl sulfoxide (DMSO, Merck, Batch K49824131817) at concentrations ranging between 0.010 and 10 mg/mL. The lower stock solutions were prepared separately by proportional dilution of the highest stock solution in DMSO. For the preparation of each test concentration, 100 µL of a stock solution was added to 1 L of test medium resulting in test concentrations that were a factor of 10,000 lower than the stock concentrations.
- Controls: Blank-control (test medium without test item or other additives) and solvent-control (test medium without test item but with 0.1 mL DMSO/L)

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Raphidocelis subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: Algae stock cultures were started by inoculating growth medium with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C.
- Pre-culture: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.
- Stock culture medium: M1, according to NPR 6505
- Pre-culture medium and test medium: M2, according to OECD201

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L (24 mg CaCO3/L)

Test temperature:

23°C

pH:

At t=0 h: 8.0-8.1
At t=72 h: 7.8

Nominal and measured concentrations:

Nominal concentrations: 0.010, 0.032, 0.10, 0.32 and 1.0 mg/L.
Measured concentrations from the stock solution in DMSO: 0.0947, 0.294, 0.374, 1.16, 8.85 mg/mL
Measured concentrations from the test samples in M2-medium: all concentrations measured at t=24 h and t=72 h were below the limit of quantification (except for the blank control at t=24 h, which showed a concentration of 6.83 mg/L of which the cause is unknown).

Details on test conditions:

TEST SYSTEM
- Test vessel: 100 mL, all-glass, capped (aluminium caps) and perforated for ventilation, containing 50 mL of test solution
- Initial cells density: 1 x 10^4 cells/mL
- Control end cells density: 129 x 10^4 cells/mL (blank control) and 125 x 10^4 cells/mL (solvent control)
- 3 replicates of each test concentration,
- 6 replicates of the control,
- 1 extra replicates of each test group for sampling purposes;
- 1 or 2 replicates of each test concentration without algae.
- Capped vessels were distributed at random in the incubator and daily repositioned. During incubation the algal cells were kept in suspension by continuous shaking.

GROWTH MEDIUM
- Standard medium used: yes, M2

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2, formulated using tap-water purified by reverse osmosis.
- Culture medium different from test medium: yes, M1
- Intervals of water quality measurement: pH: at the beginning and at the end of the test. Temperature of medium: continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod, light intensity and quality: Continuously using TLD-lamps with a light intensity within the range of 90 to 96 µE.m-2.s-1

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (path length = 10 mm). Algal medium was used as blank and the extra replicates, without algae, as background for the treated solutions.
- Other: At the end of the final test microscopic observations were performed on the 0.32 mg/L test concentration and the controls to observe for any abnormal appearance of the algae.

LIMIT/RANGE-FINDING TEST:
- A range-finding study was performed using WAFs prepared at loading rates of 1.0, 10 and 100% of an SS prepared at 100 mg/L. Results showed that growth was inhibited by 0.3% in the two lower test concentrations and 69.3% in the highest test concentration.
- In the limit/range-finding study the test item was not spiked with DMSO.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (July 2018)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95%-CI: 0.33 - 0.63 mg/L.

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: based on statistical significance

Details on results:

Analytical results:
- Stock solutions in DMSO: measured concentrations were at the level of nominal in 0.10, 0.32 and 10 mg/mL (89-95%), or at 36-37% of nominal at 1.0 and 3.2 mg/mL. To confirm the results, samples taken from 1.0 and 3.2 mg/mL were reanalysed, showing similar measured concentrations. The reason why measured concentrations were not at the level of nominal might be related to an intrinsic difficulty in collecting a homogeneous sample. The results, however, showed that the test item was present in the stock solutions at increasing concentrations and, as all were diluted starting from the highest stock (10 mg/mL) that showed a correct concentration, it was expected that all stocks contained close to nominal concentrations.
- Test samples in M2-medium: samples taken from all test concentrations and the controls were analysed. Measured concentrations were below the limit of quantification already at the start of the test, with the exception of measured concentrations of 0.69 mg/L at the highest test concentration at the start of the test. It should be noted that a response below the limit of quantification was also measured in the both controls. Additionally, a concentration of 6.8 mg/L was analysed in the blank control at 24 hours of exposure. The cause is unknown. This was, however, considered unrealistic, since the measured concentration was much higher than the highest concentration tested.

Effects parameters were based on nominal concentrations as it was expected that the volume of stock solutions used for spiking did contain the correct amount of test item making up the reported nominal concentrations.

Results of the final test:
- Statistically significant growth rate inhibition of respectively 11% and 100% was recorded at nominal concentrations of 0.32 and 1.0 mg/L at the end of the test. The 11% inhibition was not statistically significant but biologically relevant.
- No significant difference was present between the blank and solvent control and, therefore, measurements of both controls were pooled.
- Abnormalities observed: no, microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to 0.32 mg/L when compared to the control.
- The test conditions were within the limits prescribed by the study plan and the test guidelines.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Test concentrations: 0, 0.18, 0.32, 0.56, 1.0, 1.8 and 3.2 mg/L
- EC50 for growth rate inhibition (72h-ERC50) was 0.90 mg/L with a 95% confidence interval ranging from 0.88 to 0.93 mg/L.
- The observed 72h-ERC50 for the algal culture tested corresponds with the historical range of the test facility.

Reported statistics and error estimates:

An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the pooled controls revealed significant inhibition of growth rate or inhibition of yield (Step-down Jonckheere-Terpstra Test Procedure, α=0.05, one-sided, smaller).
Calculation of ECx-values was based on Weibull analysis using linear max. likelihood regression with the percentages of growth rate inhibition and the percentages of yield inhibition versus the logarithms of the corresponding nominal concentrations of the test item.
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analysis.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

see 'overall remarks'

Conclusions:

Based on an algae toxicity study, performed according to OECD guideline 201 and GLP, the 72h-EC50 for growth rate inhibition of INCA 460: MXDA Adduct towards Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) was 0.46 mg/L (95% confidence interval ranging from 0.33 to 0.63 mg/L).