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

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:

23 March 2018 - 23 July 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 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

13 April 2004

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.
- The water solubility at 20°C was determined to be <1 mg/L using the flask method.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

For determination of test concentrations in the final test, single samples for possible analysis were taken from additional vessels without organisms from all test concentrations and the control according to the schedule below.
Frequency: at t=0 h and t=48 h
Volume: 20 mL from the approximate centre of the test vessels
Storage: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the test facility

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION:
- Method: for the combined limit/range-finding test, test solutions were prepared from a saturated solution prepared at a loading rate of 100 mg/L. However, information provided after the performance of the combined limit/range-finding test revealed that the test item is a complex mixture instead of a pure substance. Therefore it was decided to prepare the test solutions for the the final test as individual WAFs. Preparation of test solutions started with loading rates individually prepared ranging between 4.6 and 100 mg/L. A 1-day period of magnetic stirring was applied to ensure maximum dissolution of the test item in medium. The obtained mixtures were allowed to settle for approximately 1 day. Thereafter, the aqueous Water Accommodated Fractions (WAFs) were collected by means of siphoning and used as test concentrations. All test solutions were clear and colorless at the end of the preparation procedure.
- Controls: Test medium without test item or other additives.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Straus, 1820
- Source: in-house laboratory culture with a known history. At least third generation, obtained by a cyclical parthenogenesis under specified breeding conditions.
- Age of parental stock: Daphnids originated from a healthy stock, 2nd to 5th brood, showing no signs of stress such as mortality >20%, presence of males, ephippia or discoloured animals and there was no delay in the production of the first brood.
- Age at study initiation: < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: no

ACCLIMATION
- Acclimation period: no

BREEDING:
- Method: each batch was started with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium (M7) in an all-glass culture vessel. Cultures were max. 4 weeks old. After 7 days of cultivation half of the medium was renewed twice a week.
- Feeding: daily, a suspension of fresh water algae.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L

Test temperature:

20°C

pH:

At t=0 h: 8.0-8.2
At t=48 h: 8.0-8.1

Dissolved oxygen:

At t=0 h: 8.1-8.8 mg/L
At t=48 h: 8.5-8.9 mg/L

Nominal and measured concentrations:

Nominal concentration: WAFs prepared at 4.6, 10, 22, 46 and 100 mg/L
Measured concentration at both t=0 and t=48 h were below the limit of quantification for WAFs prepared at 4.6, 10, 22 and 46 mg/L. The measured concentrations in the WAF prepared at 100 mg/L were 0.711 mg/L and 0.716 mg/L at t=0 and t=48 h, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: 60 mL, all-glass
- Fill volume: 50 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Standard medium used: yes, according to OECD 202
- Source/preparation of dilution water: tap water purified by reverse osmosis
- Culture medium different from test medium: yes, M7 was used as culture medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: the test was performed in the dark

EFFECT PARAMETERS MEASURED: mobility at 24 and 48 hours.
- Additional measurements: pH and dissolved oxygen at the beginning and at the end of the test, in the limit test concentration and the control. Temperature of the medium: continuously in a temperature control vessel.

COMBINED LIMIT/RANGE-FINDING STUDY: yes, twenty daphnids per concentration (four replicates, 5 daphnids per vessel) were exposed to a control and test concentrations of 1.0, 10 and 100% of the SS prepared at 100 mg/L. At 100% of the SS prepared at 100 mg/L, 10% and 30% of the introduced daphnids became immobilized at t=24 and t=48 h, respectively. A final test was used to confirm the results as interference with the spectrophotometric analysis may have occurred (test samples for test concentration determination were taken from the test system including daphnis which could have interfered).

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (July 2018)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 0.71 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mobility

Details on results:

Results of test sample analysis (see table 1):
- Recovery of the quality control at the level of 0.5 mg/L was lower than acceptable. However, since the measured concentration of the final test were comparable to those of the combined limit/range-finding test and in line with the water solubility (<1 mg/L), it was considered to have no effect on the results of the analysis.
- The measured concentrations were below the limit of quantification up to and including a WAF prepared at a loading rate of 46 mg/L.
- The measured concentrations in the WAF prepared at the highest loading rate remained constant over the test period (i.e. 0.71 and 0.72 mg/L at t=0 and t=48 h, respectively) and therefore the effect concentration was based on the initially measured concentration.

Results of the final test:
- No immobility was observed in the control and the three lowest test concentrations while 5 and 30% immobility was observed at the two highest test concentrations (see table 2). Based on these observations, the EC50 was determined to exceed the highest concentration tested.
- Abnormal observations: no
- Mortality of control: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, microscopic observation revealed no test item attached to the daphnids.
- Effect concentrations exceeding solubility of substance in test medium: yes
- The experimental conditions remained within the limits prescribed by the study plan and were according to the OECD guideline.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 48h-EC50: 0.59 mg/L, 95%-confidence interval: 0.53-0.72 mg/L
- Other: results fell within the historical range of the test facility.

Any other information on results incl. tables

Table 1 Measured concentrations in the final test

Time of sampling [hours]	Percentage of SS^*[%]	Abs. [AU]	Abs. Cor^#[AU]	Analyzed concentration [mg/L]	Relative to initial [%]
n.a.	blank	0.0095		n.d.
	blank	0.0098		n.d.
	blank	0.0099		n.d.
0	0	0.0070	-0.0027	< 0.5
	4.6	0.0113	0.0016	< 0.5
	10	0.0111	0.0014	< 0.5
	22	0.0155	0.0058	< 0.5
	46	0.0233	0.0136	< 0.5
	100	0.0306	0.0209	0.711
48	0	0.0060	-0.0037	< 0.5	n.a.
	4.6	0.0091	-0.0006	< 0.5	n.a.
	10	0.0105	0.0008	< 0.5	n.a.
	22	0.0101	0.0004	< 0.5	n.a.
	46	0.0142	0.0045	< 0.5	n.a.
	100	0.0308	0.0211	0.716	101

^*     Percentage of a saturated solution (SS) prepared at a loading rate of 100 mg/L.

^#     Absorbance of the samples is corrected for the mean of the blank i.e. 0.0097 AU (n=3).

n.d. Not detected.

n.a. Not applicable.

Table 2 Immobility observed during the final test

Time (h)	Replicate	INCA 460: MXDA Adduct Loading rate (mg/L)
		Control	4.6	10	22	46	100 (0.71)
0	A	5	5	5	5	5	5
	B	5	5	5	5	5	5
	C	5	5	5	5	5	5
	D	5	5	5	5	5	5
	Total introduced	20	20	20	20	20	20
24	A	0	0	0	0	0	1
	B	0	0	0	0	0	0
	C	0	0	0	0	0	2
	D	0	0	0	0	0	0
	Total immobilised	0	0	0	0	0	3
	Effect %	0	0	0	0	0	15
48	A	0	0	0	0	0	2
	B	0	0	0	0	0	0
	C	0	0	0	0	0	3^#
	D	0	0	0	0	1	1
	Total immobilised	0	0	0	0	1	6
	Effect %	0	0	0	0	5	30

^# Microscopic observation revealed no test item attached to the daphnids.

() – initially measured concentration (mg/L)

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

1). In the control, no daphnids became immobilised or showed other signs of disease or stress. 2). The oxygen concentration at the end of the test was ≥ 3 mg/L in control and test vessels (i.e., ≥ 8.5 mg/L).

Conclusions:

The 48h-EC50 for Daphnia magna exposed to INCA 460: MXDA Adduct exceeded the maximum solubility of the test item in test medium, i.e. exceeded a measured concentration of 0.71 mg/L.