N-(1,3-dimethylbutylidene)-5-[(1,3-dimethylbutylidene)amino]-1,3,3-trimethylcyclohexanemethylamine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23/01/2020-17/08/2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

GLP compliance:

yes

Specific details on test material used for the study:

Information as provided by the Sponsor. A Certificate of Analysis supplied by the Sponsor is
given in Annex 1.
Identification: CAS 71077-09-3
Batch: 12846
CAS Number: 71077-09-3
Purity: 97.32%
Physical State/Appearance: Pale yellow viscous liquid
Expiry Date: 30 November 2020
Storage Conditions: Room temperature in the dark, under nitrogen

Analytical monitoring:

not specified

Details on sampling:

Chemical analysis of the fresh test preparations at 0 hours (see Annex 5) showed measured
concentrations of Isophoronediamine to range from 0.64 to 48 mg/L. Analysis of the
expired/old test preparations at 72 hour showed measured concentrations of
Isophoronediamine to range from 0.72 to 50 mg/L.
The dissolved test item may have been one or several components of the test item. Given that
toxicity cannot be attributed to a single component or mixture of components but to the test
item as a whole, the results were based on nominal loading rates and at the request of the
Sponsor 0-Hour measured concentrations of Isophoronediamine.

Vehicle:

no

Details on test solutions:

Nominal amounts of test item (10, 32, 20, 64 and 200 mg) were each separately added to the
surface of 10, 10, 2.0, 2.0 and 2.0 liters of culture medium to give the 1.0, 3.2, 10, 32 and
100 mg/L loading rates respectively. After the addition of the test item, the culture medium
was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a
dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed
to stand for 1 hour. A 10 and 2.0 mL pipette, covered at one end with Nescofilm was
submerged into the vessels, sealed end down to mid-depth within the vessels. A length of
Tygon tubing was attached to the 10 mL pipette and the Nescofilm was removed using the
2.0 ml pipette. The aqueous phase or WAF was removed by mid-depth siphoning (the first
75 to 100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs.
Microscopic inspection of the WAFs showed no micro-dispersions of test item to be present.
An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal
suspension (2.9 mL) to give an initial nominal cell density of 5.00 x 103 cells/mL.
The concentration and stability of the test item in the test preparations were verified by
chemical analysis at 0 and 72 hours (see Annex 5).

Test organisms (species):

other: Raphidocelis subcapitata

Details on test organisms:

The test was carried out using Raphidocelis subcapitata strain CCAP 278/4. Liquid cultures
of Raphidocelis subcapitata were obtained from the Culture Collection of Algae and
Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll,
Scotland.
Approximately 3 to 4 days before the start of the test, inoculum cultures of algae was set up
at an initial cell density of approximately 103 cells/mL. The flasks were plugged with
polyurethane foam stoppers and kept under constant agitation by orbital shaker
(approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density
was approximately 105 to 106 cells/mL.
A positive control test using potassium dichromate as the reference item was performed twice
in a 12 month period to demonstrate satisfactory conditions of the test. Details of the most
recent positive control (Covance Study Number YY61NS) are given in Annex 2.

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

ca. 13 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Raphidocelis subcapitata based on nominal loading rates gave the following
results:
Response
Variable
EL50
(mg/L Loading
Rate WAF)
95% Confidence Limits
(mg/L Loading Rate
WAF)
No Observed
Effect Loading
Rate (NOEL)
(mg/L Loading
Rate WAF)
Lowest
Observed Effect
Loading Rate
(LOEL)
(mg/L Loading
Rate WAF)
Growth Rate 26 25 - 27 3.2 10
Yield 8.5 7.7 - 9.3 3.2 10
Exposure of Raphidocelis subcapitata based on 0-Hour measured concentrations of
Isophoronediamine gave the following results:
Response
Variable
EL50
(mg/L)
95% Confidence Limits
(mg/L)
No Observed
Effect Loading
Rate (NOEL)
(mg/L)
Lowest
Observed Effect
Loading Rate
(LOEL)
(mg/L)
Growth Rate 13 12 - 13 1.6 5.1
Yield 4.3 3.9 - 4.7 1.6 5.1

Executive summary:

1.1 Introduction

A study was performed to assess the effect of the test item on the growth of the green alga

Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata). The method

followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals

(2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method

C.3 of Commission Regulation (EC) 761/2009.

1.2 Methods

Due to the low aqueous solubility and complex nature of the test item for the purposes of the

study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test

item.

Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to Water

Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of

1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under

constant illumination and shaking at a temperature of 24 ±1 °C.

Samples of the algal populations were removed daily and cell concentrations determined for

each control and treatment group, using a Coulter® Multisizer Particle Counter.

1.3 Results

Information provided by the Sponsor indicated that the test item degraded within 2 hours and

as a results of the method of preparation for the test samples, chemical analysis was

performed on a degradant product instead of the parent material. The chemical analysis was

performed on Isophoronediamine, one of the identified degradant products of the parent

material.

Chemical analysis of the fresh test preparations at 0 hours showed measured test

concentrations of Isophoronediamine to range from 0.64 to 48 mg/L. Chemical analysis of

the aged to test preparations at 72 hours showed measured test concentrations of

Isophoronediamine to range from 0.72 to 50 mg/L.

Given that the toxicity cannot be attributed to a single component or a mixture of components

but to the test item as a whole, the results were based on nominal loading rates and at the

request of the Sponsor results were also based on the 0-Hour measured concentrations of

Isophoronediamine.

Exposure of Raphidocelis subcapitata based on nominal loading rates gave the following

results:

Response

Variable

EL50

(mg/L Loading

Rate WAF)

95% Confidence Limits

(mg/L Loading Rate

WAF)

No Observed

Effect Loading

Rate (NOEL)

(mg/L Loading

Rate WAF)

Lowest

Observed Effect

Loading Rate

(LOEL)

(mg/L Loading

Rate WAF)

Growth Rate 26 25 - 27 3.2 10

Yield 8.5 7.7 - 9.3 3.2 10

Exposure of Raphidocelis subcapitata based on 0-Hour measured concentrations of

Isophoronediamine gave the following results:

Response

Variable

EL50

(mg/L)

95% Confidence Limits

(mg/L)

No Observed

Effect Loading

Rate (NOEL)

(mg/L)

Lowest

Observed Effect

Loading Rate

(LOEL)

(mg/L)

Growth Rate 13 12 - 13 1.6 5.1

Yield 4.3 3.9 - 4.7 1.6 5.1

Key value for chemical safety assessment

EC50 for freshwater algae:

13 mg/L