2,4,6-triisopropyl-m-phenylene diisocyanate

Bioaccumulation: aquatic / sediment

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-10 - 2020-11-02 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Due to quick hydrolysis (see cross-reference), the study is performed with TRIDA in accordance with RAAF Scenario 1 (ECHA 2017).

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 305 (Bioaccumulation in Fish: Aqueous and Dietary Exposure) -I: Aqueous Exposure Bioconcentration Fish Test

Deviations:

not applicable

Remarks:

study ongoing

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Details on sampling:

- Sampling intervals/frequency for test organisms:
For chemical analysis: At test start, 10 individuals were sampled from the stock population. At each sampling event, 4 individuals were sampled from the test item and control treatments. During the uptake phase 7 samplings were performed on Day 1, 2, 5, 8, 12, 15 and 19 of exposure.The fish were sampled before feeding.
For lipid content analysis: At the start of the uptake phase, 5 individuals were sampled from the stock population. At the end of the uptake phase, 5 individuals were sampled from the control and treatment group.
- Sampling intervals/frequency for test medium samples: Water samples of the control and the test item treated group were taken for determination of the test item concentration before addition of the fish (Day -1) and at each sampling occasion during the uptake phase (Day 1, 2, 5, 8, 12, 15 and 19 of exposure).
- Details on sampling and analysis of test organisms and test media samples (e.g. sample preparation, analytical methods):
Preparation of water samples: An aliquot of the sample was diluted by factor 20 using acetonitrile/pure water (50/50, v/v).
Extraction of fish tissue: The whole body of each individual fish was analysed separately. Acetonitrile/pure water (80/20 v/v) + 0.1 % formic acid was used as extraction solvent.
The fish was weighed into a tissue lyser and homogenised for 5 min. The homogenised tissue was transferred to a tube. The mill tube was washed with extraction solvent used for the first extraction step. The extraction in 20 mL PE tubes included in total three extraction steps: 10 mL extraction solvent, 5 min ultra-sonic treatment and 15 min shaking. After each step the extract was centrifuged (3000 rpm, 10 minutes) and the supernatant was transferred into a vial.
The supernatant (1.5 mL) was transferred to a QuEChERS SPE tube and vortex mixed for 2 min. The tube was finally centrifuged (8500 rpm, 10 minutes).
- Sample storage conditions before analysis: Water samples and fish tissue extracts will be stored in a freezer at ≤ -20 °C until analysis.

Vehicle:

yes

Remarks:

dimethylformamide (DMF)

Details on preparation of test solutions, spiked fish food or sediment:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 5 days prior test start the dosing system was set up and the test item concentration in the test water (0.5 mg/L) was verified analytically.
An appropriate amount of the test item was used to prepare a solution of 5 g/L in DMF. The stock solution was diluted to 500 mg/L with pure water. The equivalent amount of the organic solvent was then 10%.
During the uptake phase, one group of fish was exposed to the test item in treated water under continuous renewal (flow-through) conditions at a nominal concentration of 0.5 mg/L. This concentration corresponds to 1 % of the acute LC50 value (56.2 mg/L), and was below its water solubility (1.1 g/L). The selected concentration in the test water is by at least factor ten above the LOQ of the analytical method.
The dilution water and the application solutions were pumped into mixing vessels (one per replicate) with a mean flow rate of 0.112 mL/min (application solution) and 100 mL/min (test water). As such the application solutions and the dilution water were continuously mixed and were poured into the aquaria. A nominal test concentration of 0.500 mg/L resulted. The water in the tank contained 0.01% organic solvent.
Prior to the initiation of the test the dosing system was calibrated through the use of appropriate analysis techniques
- Control: In the control group, non-treated application solution with an equivalent amount of the organic solvent was used (10%). The dilution water and the solvent solutions were pumped into the mixing vessels (one per replicate) with a mean flow rate of 0.104 mL/min (solvent solution) and 100 mL/min (test water). As such the application solutions and the dilution water were continuously mixed and were poured into the aquaria. The water in the tank contained 0.01% organic solvent.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide (DMF)
- Concentration of vehicle in test medium (stock solution and final test solution(s) at different concentrations and in control(s)): The water in the control and the treatmant tank contained 0.01% organic solvent.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no

Test organisms (species):

Oncorhynchus mykiss (previous name: Salmo gairdneri)

Details on test organisms:

TEST ORGANISM
- Common name: Rainbow trout (Oncorhynchus mykiss)
- Source: The test fish were obtained from a commercial fish breeder (Aquakulturbetrieb Störk, 88348 Bad Saulgaul – Wagenhausen, Germany).
- Age at study initiation (mean and range, SD): juvenile
- Length at study initiation (length definition, mean, range and SD): The body length of a representative number of the test fish population (10 fish) were determined prior to the start of the test. The body length of the test fish was in the range of 3.6 to 4.4 cm, with a mean of 3.9 ± 0.2 cm.
- Weight at study initiation (mean and range, SD): The body weight of a representative number of the test fish population (10 fish) were determined prior to the start of the test as well. The body weight of the fish was in the range of 370 mg to 689 mg, with a mean of 460 ± 100 mg.
Some of the fish were smaller than half the weight of the largest. However, the largest can be taken as an outlier, leading to fish at most 70% smaller than the second largest (532 mg). The reason was a broad weight range of the stock population.
- Weight at termination (mean and range, SD): At the end of the uptake hase and test termination the mean weight was 539 ± 142 mg and the mean length was 4.2 ± 0.3 cm.
- Lipid content at test initiation (mean and range, SD): mean 1.3 %
- Feeding during test
- Food type: A commercially available fish food (Forellenbrutfutter INICIO PLUS G0.6) was used. The food was characterised in terms of crude protein and crude fat content and contained 61.3% crude protein and 8.4% crude fat.
- Amount: Approximately 1.25 - 1.50% of body weight per day. Initial feeding was based on the scheduled weight measurements of the stock population just prior to the start of the test. The amount of feed was adjusted based on the wet weights of sampled fish at each sampling event and to account for growth during the experiment.
- Frequency: The fish were fed daily throughout the acclimation period and the test period.

ACCLIMATION
- Acclimation period: After arrival, the stock population of fish was acclimated for 15 days. Holding conditions (water, temperature) were the same as the test conditions. Thus the holding period corresponded to the acclimation period.
- Acclimation conditions (same as test or not): After arrival, the stock population of fish was acclimated in water at the test temperature and was fed throughout on a sufficient diet. Both water and diet were of the same type as those used during the test.
- Health during acclimation (any mortality observed): Five fish died during the holding, which corresponds to less than 5% over 15 days.

Route of exposure:

aqueous

Justification for method:

aqueous exposure method used for following reason: water solubilty 1.1 mg/L and log Kow < 4

Test type:

flow-through

Water / sediment media type:

natural water: freshwater

Total exposure / uptake duration:

19 d

Hardness:

1.78 mmol/L (= 178.4 mg/L) as CaCO3

Test temperature:

15°C ± 2°C (continuously monitored)

pH:

control group: 7.5 - 8.0
treatment group: 7.7 - 7.9

Dissolved oxygen:

At least 60% of the saturation value

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass aquaria
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: approx. 40 litres test medium
- Type of flow-through (e.g. peristaltic or proportional diluter): The dilution water and the application solutions were pumped into mixing vessels (one per replicate) with a mean flow rate of 0.112 mL/min (application solution) and 100 mL/min (test water). As such the application solutions and the dilution water were continuously mixed and were poured into the aquaria.
- Renewal rate of test solution (frequency/flow rate): Five-fold per day
- No. of organisms per vessel: 75 per control and test item tretament group
- No. of vessels per concentration (replicates): 1
- No. of vessels per control / vehicle control (replicates): 1
- Biomass loading rate: The initial fish biomass to water ratio was of 0.23 g/L passing through the test unit per 24 hours. A fish-to-water loading rate of 0.1-1.0 g of fish (wet weight) per litre of water per day was respected.
- Other: The test is conducted in a controlled environment room, in a ventilated area.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water was used in the test. The detailed composition is given in section "Any other information on materials and methods incl. tables".
- Holding medium different from test medium: Holding conditions (water, temperature) were the same as the test conditions

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark
- Light intensity: 416 to 786 lux

Nominal and measured concentrations:

nominal: 500 µg/L and a control
time weighted average: 479.6 µg/L and a control

Reference substance (positive control):

no

Details on estimation of bioconcentration:

BASIS INFORMATION
- Measured/calculated logPow: 3.8

Lipid content:

1.3 %

Time point:

start of exposure

Lipid content:

>= 1.6 - <= 1.7 %

Time point:

end of exposure

Lipid content:

1.5 %

Time point:

other: overall mean value used for lipid-normalisation

Key result

Conc. / dose:

500 µg/L

Temp.:

>= 13 - <= 17 °C

Type:

other: BCFSSL (bioconcentration factor for steady-state including normalisation to 5% lipid content)

Value:

>= 4.5 - <= 6.1 L/kg

Basis:

normalised lipid fraction

Calculation basis:

steady state

Remarks on result:

other:

Remarks:

Based on the very low uptake observed, no depuration phase was performed and no depuration was determinable.

Rate constant:

growth rate constant (d-1)

Remarks on result:

other:

Remarks:

Because no significant bioaccumulation in fish was detectable and no BCFK was calculated, no growth rate correction was required and therefore, no growth rate constant was calculated

Details on kinetic parameters:

As no depuration phase was performed, no kinetic BCF (BCFk), uptake rate (k1) depuration rate (k2) constants, half-life (t½) and growth corrected half-life (t½g) were calculated.

Details on results:

- Mortality of test organisms: Two fish died in the test item treated group during the test (< 10% mortality).
- Behavioural abnormalities: No behavioural abnormalities were observed.
- Observations on body length and weight: At each sampling occasion the weight and lengths of the sampled fish was determined. On the day of application, the mean weight was 460 ± 100 mg and the mean length was 3.9 ± 0.2 cm. At the end of the uptake phase, the mean weight was 539 ± 142 mg and the mean length was 4.2 ± 0.3 cm.
- Mortality and/or behavioural abnormalities of control: Seven fish died in the control group during the test (< 10% mortality) and no behavioural abnormalities were observed.

Validity criteria fulfilled:

yes

Conclusions:

Due to the fast hydrolysis of the registered substance (see Neuland 2020) the bioaccumulative potential was analysed for the main hydrolysis product. The study was running under GLP according to OECD TG 305-I using aqueous exosure. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines. Hence, the results can be considered as reliable to assess the bioaccumulation potential of the test substance towards fish.
The study was running with Rainbow trout (Oncorhynchus mykiss) as test organism exposed to a single concentration of 500 µg test material/L and a control. During the test, mortality was < 10% in the control and the treatment group. No behavioural abnormalities were observed. The mean lipid content of the fish were 1.3% (start uptake phase) and 1.6-1.7% (end uptake phase). The overall mean value (1.5%) was used for lipid normalisation.
The overall mean concentration of the test material in test water during the uptake phase was 479.6 µg/L (time weighted average), representing 95.9% of the target value (500 µg/L). During the uptake phase the test item concentration was considered constant. In the control group, no test item was detectable during the uptake phase.
From day 1 to day 19 of the uptake phase the analysed concentration ranged from 0.701 to 0.921 mg/kg. An apparent plateau was therefore reached. As such, steady state calculations were based on the mean tissue concentrations from days 1 to 19 of the uptake phase. The mean measured concentrations in whole body fish tissues during the last three sampling points of the uptake phase (steady state) was 0.828 mg/kg.
Based on the very low uptake observed, no depuration phase was performed. This is in accordance with the OECD 305 guideline: “A depuration phase is always necessary unless uptake of the substance during the uptake phase has been insignificant.”
Based on the mean measured concentration of the test material in the test water, the bioconcentration factor was determined for steady state (BCFSS), including a normalisation to 5% lipid content (BCFSSL) and was 1.44-1.88 L/kg and 4.5-6.1 L/kg, respectively, indicating a low potential for bioaccumulation.

Executive summary:

The potential of the registered substance to bioaccumulate in the aquatic environment was studied according to the OECD Guidelines for Testing of Chemicals (2012) No. 305 "Bioaccumulation in Fish: Aqueous and Dietary Exposure" with Rainbow trout (Oncorhynchus mykiss) in a continuous flow-through system under GLP. Due to the fast hydrolysis of the registered substance (see Neuland, 2020) the bioaccumulative potential was analysed for the main hydrolysis product.

 

The purpose of this study was to determine the uptake, bioconcentration and elimination of the test material. The test material has a log K_OW of 3.8 and the water solubility is 1.1 g/L at the temperature of 20°C. Thus the aqueous exposure test was performed. The test initially consisted of two phases: the exposure (uptake) and post-exposure (depuration) phase. During the uptake phase (for a period of 19 days), one group of fish was exposed to the test item in treated water under continuous renewal (flow-through) conditions at a nominal concentration of 0.5 mg/L. This concentration corresponds to 1 % of the acute LC₅₀ value of the test material (56.2 mg/L), and was below its water solubility (1.1 g/L). Beside, the selected concentration in the test water is by at least factor ten above the LOQ of the analytical method. A control group, consisting of a test vessel treated only with dilution water containing the same amount organic solvent as used to set up the test medium was also included (0.01% final organic solvent concentration in the test vessels).

Based on the results of the uptake phase, no depuration phase was performed. The test was performed in aquaria containing 40 L test water with a 5-fold water exchange per day. Water and fish were sampled throughout the exposure phase and the test item concentration was determined by LC-MS/MS. Test temperature was in the range of 15 ± 2°C and the photoperiod was 16 h light : 8 h dark.

Based on the obtained results the mortality observed during the test was < 10% in the control and treatment group. No behavioural abnormalities were observed in both groups. The mean lipid content of the fish was 1.3% (start uptake phase) and 1.6-1.7% (end uptake phase). The overall mean value of 1.5% was used for lipid normalisation.

The overall mean concentration of the test material in test water during the uptake phase was 479.6 µg/L (time weighted average), representing 95.9% of the target value (500 µg/L). During the uptake phase the test item concentration was considered constant. In the control group, no test item was detectable during the uptake phase.

From day 1 to day 19 of the uptake phase the analysed concentration ranged from 0.701 to 0.921 mg/kg. An apparent plateau was therefore reached. As such, steady state calculations were based on the mean tissue concentrations from days 1 to 19 of the uptake phase. The mean measured concentrations in whole body fish tissues during the last three sampling points of the uptake phase (steady state) was 0.828 mg/kg.

Based on the mean measured concentration of the test material in the test water, the bioconcentration factor was determined for steady state (BCF_SS), including a normalisation to 5% lipid content (BCF_SSL) and was 1.44-1.88 L/kg and 4.5-6.1 L/kg, respectively, indicating a low potential for bioaccumulation.

.

Reference 2

Endpoint:

bioaccumulation in aquatic species: fish

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reference 3

Endpoint:

bioaccumulation in aquatic species: fish

Type of information:

(Q)SAR

Adequacy of study:

key study

Study period:

12 June 2020

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
Estimation Programs Interface (EPI) Suite for Microsoft Windows, v4.11 (US EPA, 2012)

2. MODEL (incl. version number)
BCFBAF v3.01

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CC(C)c1cc(C(C)C)c(N=C=O)c(C(C)C)c1N=C=O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See attached QMRF.

5. APPLICABILITY DOMAIN
See attached QPRF.

6. ADEQUACY OF THE RESULT
- The model is scientifically valid (see attached QMRF).
- See attached QPRF for reliability assessment.

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

- Software tool(s) used including version: EpiSuite v4.11
- Model(s) used: BCFBAF v3.01
- Model description: see field ''Attached justification'
- Justification of QSAR prediction: see field 'Attached justification'

Specific details on test material used for the study:

CC(C)c1cc(C(C)C)c(N=C=O)c(C(C)C)c1N=C=O

Test organisms (species):

other: Fish, not further specified

Test type:

other: QSAR

Water / sediment media type:

natural water: freshwater

Details on estimation of bioconcentration:

Using the computer tool BCFBAFWIN v3.01 by US-EPA (EPIWIN) the Bioconcentration Factor (BCF) can be estimated. The following two different models are used: Regression-based estimate (Traditional method), based on WSKOWWIN and Arnot-Gobas method, based on mechanistic first principles.

Type:

BCF

Value:

7 065 L/kg

Basis:

whole body w.w.

Remarks on result:

other: Regression-based estimate (Traditional Method), based on WSKOWWIN

Type:

BCF

Value:

5 970 L/kg

Basis:

whole body w.w.

Remarks on result:

other: Arnot-Gobas method, based on mechanistic first principles

Details on results:

Prediction of apparent metabolism half-life in fish:
Arnot-Gobas method, upper trophic: 152.8 L/kg wet-wt
Arnot-Gobas method, mid trophic: 210.5 L/kg wet-wt
Arnot-Gobas method, lower trophic: 232.2 L/kg wet-wt

Validity criteria fulfilled:

yes

Remarks:

scientifically accepted calculation method

Conclusions:

The study report describes a scientifically accepted calculation method for the bioconcentration factor estimation using the US-EPA software BCFBAF v3.01. No GLP criteria are applicable for the usage of this tool and the QSAR estimation is easily repeatable. Using the regression-based estimate (traditional method) a BCF of 7065 L/kg wet-wt was calculated. Using the Arnot-Gobas method, which is based on the mechanistic first principles, the BCF results in a value of 5970 L/kg wet-wt. The whole body primary biotransformation rate estimate for fish gives a half-life of 3.214 days, whereby the rate constant (kM) for 10 g fish is designated as 0.2157/day. This is taken into account to predict the apparent metabolism half-life in fish for the substance. For the lower trophic level a BCF of 232.2 L/kg wet-wt is calculated, whereas for the mid trophic level the BCF will result in 210.5 L/kg wet-wt and the higher trophic level gives a value of 152.8 L/kg wet-wt.

Executive summary:

The prediction for the bioconcentration factor (BCF) of the substance 2,4,6 -triisopropyl-m-phenylene diisocyanate was determined by the computer program BCFBAF v3.01 (EPIWIN software) by US-EPA (2012). Furthermore the whole body primary biotransformation rate estimation for fish was calculated with the notation that the bio half-life normalized to 10 g fish at 15 °C. It is possible to predict the apparent metabolism half-life in fish for three different trophic levels (lower, mid and upper). Using the regression-based estimate (traditional method) a BCF of 7065 L/kg wet-wt was calculated. Using the Arnot-Gobas method, which is based on the mechanistic first principles, the BCF results in a value of 5970 L/kg wet-wt. The whole body primary biotransformation rate estimate for fish gives a half-life of 3.214 days, whereby the rate constant (kM) for 10 g fish is designated as 0.2157/day. This is taken into account to predict the apparent metabolism half-life in fish for the substance. For the lower trophic level a BCF of 232.2 L/kg wet-wt is calculated, whereas for the mid trophic level the BCF will result in 210.5 L/kg wet-wt and the higher trophic level gives a value of 152.8 L/kg wet-wt.

Description of key information

1) Key_Bioaccumulation: aquatic / sediment: BCF_SSL = 4.5 - 6.1 L/kg for Onchorhynchus mykiss (flow-through, freshwater, OECD 305 -I: Aqueous Exposure Bioconcentration Fish Test, GLP)

2) Waiver_Bioaccumulation: aquatic / sediment: According to REACH Regulation (EC) No 1907/2006 Article 2 (9), polymers are excluded from Titles II (Registration) and VI (Evaluation).

3) Supporting_Bioaccumulation: aquatic / sediment_QSAR: BCFBAF v3.01 (EPISuite v4.11, US EPA 2012), BCF = 7065 L/kg ww

Key value for chemical safety assessment

BCF (aquatic species):

6.1 L/kg ww

Additional information

Due to the very fast hydrolysis (DT₅₀ = 37s) hydrolysis and the resulting instability of the test substance, it was considered most meaningful to cover the information on the bioaccumulative potential by the consideration of the degradation products. Based on the results of the hydrolysis study conducted at CURRENTA GmbH according to OECD 111, 2,4,6-triisopropyl-m-phenylene diamine (TRIDA, CAS 6318-09-08) was expected as major hydrolysis and polymeric ureas as potential minor hydrolysis products (Neuland, 2020).

Accordingly, the bioaccumulative potential was analysed for the main hydrolysis product 2,4,6-triisopropyl-m-phenylene diamine (TRIDA, CAS 6318-09-08). The study was running under GLP according to OECD TG 305-I using aqueous exosure. Based on the mean measured concentration of the test material in the test water, the bioconcentration factor was determined for steady state (BCF_SS), including a normalisation to 5% lipid content (BCF_SSL) and was 1.44-1.88 L/kg and 4.5-6.1 L/kg, respectively, indicating a low potential for bioaccumulation. As a worst-case assumption the upper value of 6.1 L/kg is taken as key value for further risk assessment.

Polymeric ureas are considered as polymers under REACH. Therefore they do not have to be registered and evaluated according to Chapter 1; Article 2, Paragraph 9 Regulation No. 1907/2006. Regardless of the evaluation of available data, the determination of the bioaccumulative potential of the polymeric ureas as minor hydrolysis product is waived accordingly.

As supporting information the bioconcentration factor (BCF) of the test substance was determined by the computer program BCFBAF v3.01 (EPIWIN software) by US-EPA (2012). Furthermore the whole body primary biotransformation rate estimation for fish was calculated with the notation that the bio half-life normalized to 10 g fish at 15 °C. It is possible to predict the apparent metabolism half-life in fish for three different trophic levels (lower, mid and upper). Using the regression-based estimate (traditional method) a BCF of 7065 L/kg wet-wt was calculated. Using the Arnot-Gobas method, which is based on the mechanistic first principles, the BCF results in a value of 5970 L/kg wet-wt. The whole body primary biotransformation rate estimate for fish gives a half-life of 3.214 days, whereby the rate constant (kM) for 10 g fish is designated as 0.2157/day. This is taken into account to predict the apparent metabolism half-life in fish for the substance. For the lower trophic level a BCF of 232.2 L/kg wet-wt is calculated, whereas for the mid trophic level the BCF will result in 210.5 L/kg wet-wt and the higher trophic level gives a value of 152.8 L/kg wet-wt. No GLP criteria are applicable for the usage of this tool, but due to the fact that it is a scientifically accepted calculation method the estimations performed are reliable with restrictions.