Reaction products of N6,N6'-hexane-1,6-diylbis[N2,N4-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4,6-triamine] and allylbromide, subsequently reacted with ethaneperoxoic acid, hydrogenated

Toxicity to terrestrial arthropods

Administrative data

Endpoint:

toxicity to terrestrial arthropods: long-term

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 JUN 2019 - 15 MAY 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Synonym: GSID 3056-2; (2,2,6,6, tetramethyl-4-piperidinyl)-1,3,5-triazine-2,4,6-triamine, hydrogenated
Batch no: nd694_ME14
Test substance no.: 16/0020-1
Content: Total content of components of the given chemical identity is assumed as > 99 g/100 g (UVCB), water as 0.06 g/100 g and side product Toluene as 0.06 g/100 g
Homogeneity: homogeneous
Molar mass: 1742.7 g/mol
Date of production: 02 Nov 2015
Physical state/Appearance: solid/light pink or rose-colored powder
Water solubility: 1 mg/L with 0.4 g/L loading; 3 mg/L with 4 g/L loading
Other solubility: The substance is soluble in organic solvents such as methylethylketone. It is poorly soluble in ethanol
Log Pow: >5 at 23 °C
Storage conditions: ambient (room temperature)
Storage stability: The stability under storage conditions over the test period is guaranteed by the sponsor, and the sponsor holds this responsibility. The test facility is organizationally independent from the BASF SE sponsor division.
Expiry date: 02 Nov 2020

Analytical monitoring:

not required

Details on preparation and application of test substrate:

TEST SUBSTRATE
The earthworms were tested in an artificial soil. The composition of this soil was based on OECD Guideline No. 222 (the percentages refer to the dry weight):

33.3 kg quartz sand (74% of total substrate mass), Supplier: ISP Industrie Sandwerke Pfalz GmbH & Co.KG, 67240, Germany
9.5 kg kaolin clay (21% of total substrate mass), Supplier: Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany
2.3 kg sphagnum peat (5% of total substrate mass), finely grounded, Supplier: Raiffeisen Mannheim eG, 68307 Mannheim, Germany
110.1 g CaCO3, p.A. (0.2% of total substrate mass), Supplier: Merck

Total mass of the test substrate: 45.21 kg
Water holding capacitymax: 47.4 g/100g dw (mean value of two single determinations: 49.1/45.7)

The availability of lipophilic test item (ingredients) in soil and therefore their toxicity to earthworms depends on the organic matter content of the soil. For chemicals with logKow >2, adsorption is expected to be linearly related to soil organic matter content. With respect to the properties of the test item (log Kow >2), 5% of peat instead of 10% were used, considering the influence on bioavailability (OECD 222 and EPPO 2003).

TEST ITEM PREPARATION
The test substance is a solid material. The water solubility is not enough to prepare a sufficient stock solution with deionized water. Therefore, the test substance was mixed with quartz sand for the application. For this, required amounts of test substance was added to 25 g quartz sand and thoroughly mixed (see table 1 below). This test substance- quartz sand mix was then added to 2500 g (dry weight) of prepared test substrate. The amount of soil required for 8 replicates of control was 5 kg and was beyond the maximum capacity of the tray used for mixing the test substrate. Therefore, the control test assays were prepared in two portions of 2.5 kg. The test mixture of the control contained only quartz sand without test substance.

PREPARATION OF THE TEST MIXTURES WITH TEST SUBSTANCE
The required amounts of artificial soil were weighed into a mixing tray. The test substance / quartz sand mixtures were added and thoroughly mixed with the soil. Thereafter, the water content of the soil was adjusted to about 50% WHCmax using deionized water and was mixed once again. The test mixtures were distributed to the test units.

Each replicate container was filled with about 680 g of the prepared test mixture (corresponding to about 550 g Dw artificial soil) to a layer of about 4 cm and a corresponding surface area of about 170 cm².

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Breeding facility: BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
- Age at test initiation (mean and range, SD): about 12 months
- Weight at test initiation (mean and range, SD): fresh weight between 300 and 600 mg
- only adult worms with clitellum were used

ACCLIMATION
Before starting the test, the worms selected were acclimatized in the test substrate in a plastic box under test conditions through overnight under permanent light and without feeding. For this, a plastic box was filled up with approx. 750 g test substrate (dw) of which the water content is adjusted to 50 ±10% WHCmax and added with a sufficient number of adult worms that were sorted out from the breeding boxes.

Study type:

laboratory study

Substrate type:

artificial soil

Remarks:

5% sphagnum peat

Limit test:

no

Total exposure duration:

56 d

Test temperature:

20.0 - 21.9 °C

In a separate vessel, filled with about 750 g test substrate (dry weight), around two times per week during the exposure time with an electronic thermometer. The water content of this vessel was adjusted initially (on day 0) to the water content of test vessels.

pH:

6.4-6.5. At the start and end of exposure, an amount of 6.2 g wet test substrate was weighed in a beaker. Then 25 mL of 0.01 molar calcium chloride solution was added to this beaker. These mixtures were stirred for 5 minutes and rested for 2 hours. The pH values were then measured in the supernatant of these samples.

Moisture:

47.7 - 61.4% WHCmax.

A higher value of water content above the required criteria of 50 ±10% WHCmax was observed, probably due to the excessive spraying of deionised water during the moisturisation of food. However, an effect to the test results can be excluded.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): Plastic dishes with a volume of about 1.0 L, dimensions 11×15.5×6 cm with transparent and punctured lid
- Amount of soil or substrate: approx. 680 g artificial soil (wet weight)
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

OTHER TEST CONDITIONS
- Photoperiod: 16 h light and 8 h dark
- Light intensity: 514 lux (mean of 5 single measurements: 792, 438, 419, 507 and 412 lux)
- Feeding: 5 g finely ground cow manure + 5-6 mL deionized water on the test substrate surface on day 1, 7, 14 and 21. On day 28, the food was mixed with the test substrate of each test vessel.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : number of adult worms and biomass at start of expore and after 28 days, number of juvenile worms at the end of exposure (56d), consumption of food and abnormalities or behavioural changes were reported weekly (day 1, 7, 14, 21, 28, 35, 42, 49, 56).

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Test concentrations: 62.5, 125, 250, 500 and 1000 mg/kg dw (nominal)

Nominal and measured concentrations:

62.5, 125, 250, 500 and 1000 mg/kg dw (nominal)

Reference substance (positive control):

yes

Remarks:

BAS 346 F (Carbendazim) was tested as reference substance in order to show the sensitivity of the test system by a non-GLP project 68E0902/00G027 (start of exposure 15 April 2019).

Key result

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

Biomass change

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

No mortality of adult worms was observed in the controls and in all replicates with test substance. No ECx calculations were performed, because there was no dose response relationship. No abnormalities or behavioural changes were observed during the whole test duration.

Results with reference substance (positive control):

BAS 346 F (Carbendazim) was tested as reference substance in order to show the sensitivity of the test system by a non-GLP project 68E0902/00G027 (start of exposure 15 April 2019). The raw data and the report are archived.

ECx values [mg/kg soil (dw)], reproduction by counting juvenile worms, conf. limits 95%:
EC10: 0.56, LCL: 0.45, UCL: 0.71
EC50: 1.03, LCL: 0.91, UCL: 1.16

These observed effects show a high sensitivity of the test system and the derived EC50 value for reproduction toxicity by counting the number of juvenile worms is within the expected range from the test guideline (1-5 mg/kg carbendazim/kg soil (dw)) and hence acceptable sensitivity of the test system is assured.

Reported statistics and error estimates:

The statistical calculations to determine the NOEC/LOEC were carried out using SAS
System computer software.

For the changes in body weight of live adults (% of initial weight) after the first 4
weeks the statistical evaluation was carried out using Williams trend test. This test
was not significant and therefore the Dunnett's test for a simultaneous comparison
of several dose groups with the control was carried out. The statistical unit was the
test container. The tests were performed one-sided.

For the number of juveniles (offsprings) the Jonkheere-Terpstra trend test
was performed. Since this test is not significant, a pair-wise comparison of each dose
group with the control was carried out using Wilcoxon-test. The statistical unit was
the test container. The tests were performed one-sided.

The trend tests for the changes in body weight of live adults and for the number
of juveniles showed no significant result.

No ECx calculations were performed, because there was no dose
response relationship.

Validity criteria fulfilled:

yes

Conclusions:

No effects could be observed towards Eisenia fetida. The NOEC for mortality, biomass change and reproduction were ≥1000 mg/kg d.w., and, accordingly, the LOEC >1000 mg/kg d.w.. No ECx calculations were performed, because there was no dose response relationship. 

Executive summary:

The toxicity to terrestrial macroorganisms was determined in a study according to OECD guideline No 222 and in compliance with GLP criteria.

A 56-day reproduction test with Eisenia fetida was performed. Test concentrations of 62.5, 125, 250, 500 and 1000 mg/kg d.w. (dry weight) were tested. The following endpoints were recorded:

·Biomass of adult animals: day 0 and 28

·Mortality of adult worms: day 28

·Reproduction: day 56

The test met the acceptability criteria prescribed by the study plan and were considered valid. Under the conditions of the present study, the substance did not have any adverse effects on mortality, biomass change nor reproduction. The NOEC for mortality, biomass change and reproduction was ≥1000 mg/kg d.w., and accordingly, the LOEC >1000 mg/kg d.w.. No EC_x calculations were performed, because there was no dose response relationship.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

2.5208g of the test substances was dissolved in 10ml dichloromethane to give the solvent stock solution. The solvent stock solution was inverted several times to ensure adequate mixing and homogeneity. An aliquot solvent solution was separately added to a certain amount of silica sand and the solvent allowed to evaporate off prior to incorporation into formulated artificial soil with deionised reverse osmosis water. These were mixed by a mixer to give the blank control, solvent control, 1000mg/kg respectively with a nominal moisture content of 40-60% by mass of the total water-holding capacity with the deionized water. The test medium preparation process of the blank control group was same as the other groups, but without the solvent and the test substance. The test medium preparation process of the solvent control group was same as the other groups, but without the test substance.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

The study was performed with earthworms (Eisenia fetida). This species was recommended in "Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals- Effects on Biotic Systems, 2013, 207 Earthworm, Acute Toxicity Test" and "OECD, Guidelines for the Testing of Chemicals, 1984, 207 Earthworm, Acute Toxicity Test". The earthworms were purchased from Jiang Su province Jurong Stock Farm (ID: EFMKT- 160918) in the limit test. All newly acquired earthworms were quarantined and observed for 14 days prior to use in the test. Earthworms are bred on a substrate consisting of 50% by volume peat and 50% by volume cow manure and kept under 20 ± 2°c for 7 days prior to testing. The test organisms were with clitellum and were transferred into artificial soil (without test substance) approximately 24 hours prior to the start of the test. At the start of the test, the adult earthworms used originated from the same population, were with clitellum and wet weight of 300 to 600mg. The LC50 of the toxic reference substance chloroacetamide was determined once a year following "Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals- Effects on Biotic Systems, 2013, 207 Earthworm, Acute Toxicity Test" and "OECD, Guidelines for the Testing of Chemicals, 1984, 207 Earthworm, Acute Toxicity Test". The most recent test with chloroacetamide was performed from 28th Sep. 2016 to 11th Oct. 2016 (14 days test). In this study, the reference substance was tested at five concentrations of 3.75, 7.50, 15, 30 and 60mg/kg dry artificial soil weight. The mortalities of chloroacetamide treatment groups were shown to be between 0% to 100%. The 14- days LC50 for chloroacetamide was 21.4mg/kg dry soil weight. Test from this reference substance conducted during 2009 and 2015 in the laboratory and show that the range of LC50 is between 3.1 to 30.3mg/kg, so the 21.4mg/kg was an acceptable value.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

yes

Total exposure duration:

14 d

Test temperature:

20.0 - 20.3 deg. C

pH:

6.38 - 6.48

Moisture:

25.19 - 27.23% (expresed as % dry weight)

Details on test conditions:

For the controls and the treatments, 10 earthworms were added to each 1 liter closed glass vessel with riser vent containing approximately 750g wet weight of the substrate. The vessels were covered and maintained under continuous illumination (608-635Iux) in a climate chamber at 20.0 to 20.3°C for a period of 14 days. The earthworms received no food during exposure. The total gross weights of the earthworms added to each vessel in the controls and the treatments were recorded on Day 0 before addition. The surviving earthworms in each vessel were weighed on Day 14. Mortalities were recorded at 7 and 14 days after the start of test.

Nominal and measured concentrations:

nominal: 1000 mg/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

chloro acetamide

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Validity criteria fulfilled:

yes