3,5,5-trimethylcyclohex-2-enone

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

fish adult: (sub)lethal effects

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2020

Reference 2

Reference Type:

other company data

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD TG 229 (Fish Short Term Reproduction Assay)

Version / remarks:

2009

Deviations:

yes

Remarks:

These deficiencies/deviations do not have an impact on the interpretation of this study.

GLP compliance:

no

Remarks:

Signed and dated Quality Assurance claims statements were provided. A signed and dated GLP statement were not provided and appear to have been inadvertently omitted.

Test material

Test material information

Constituent 1

Reference substance name:

3,5,5-trimethylcyclohex-2-enone

EC Number:

201-126-0

EC Name:

3,5,5-trimethylcyclohex-2-enone

Cas Number:

78-59-1

Molecular formula:

C9H14O

IUPAC Name:

3,5,5-trimethylcyclohex-2-enone

Test material form:

liquid

Details on test material:

- State of aggregation: Liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Lot No.: 1136041301
- Purity: 98.7%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: stable

Sampling and analysis

Analytical monitoring:

yes

Test solutions

Vehicle:

no

Test organisms

Aquatic vertebrate type:

fish

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead Minnow
- Source: A single laboratory stock. New England Bioassay, Inc., Manchester, Connecticut
- Life stage:
- Age at study initiation (mean and range, SD): 6 months
- Length at study initiation (length definition, mean, range and SD): not reported
- Weight at study initiation (mean and range, SD): 3.7 g (males), 1.8 g (females)
- Pre-exposure reproductive information >= 15 eggs / female / reproductive day / replicate

ACCLIMATION
- Acclimation period: 28 days
- Acclimation conditions (same as test or not): same
- Type and amount of food during acclimation: frozen brine shrimp (2.0 - 2.5 mL frozen adult brine shrimp, thawed)
- Feeding frequency during acclimation: 2 times /day
- Health during acclimation (any mortality observed): no mortalities was observed

FEEDING DURING TEST
- Food type: frozen brine shrimp
- Amount: 2.0 - 2.5 mL frozen adult brine shrimp, thawed)
- Frequency: 2 times /day

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs):
- Method of collection of fertilised eggs:
- Subsequent handling of eggs:
- Subsequent handling of juvenile fish:

POST-HATCH FEEDING
- Start date:
- Type/source of feed:
- Amount given:
- Frequency of feeding:

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Hardness:

Mean: 68 (64 - 72) mg/l as CaCO3

Test temperature:

Min: 24.6 °C
Max: 25.4 °C
Mean: 25 °C

pH:

7.0 - 7.5
Mean: 7.3

Dissolved oxygen:

Mean: 7.4 mg/L ( 6.8 - 7.9 mg/L)

Conductivity:

198 µmhos/cm

Nominal and measured concentrations:

Nominal: Cotrol, 0.8, 8 and 80 mg/l
Mean measured: Control, 0.748, 7.62 and 79.8 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: glass aquaria with silicone adhesive
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 20 x 39 x 25 cm, solution depth - 13 cm / fill volume: 10 L
- Aeration: no
- Type of flow-through (e.g. peristaltic or proportional diluter): continuous-flow proportional diluter
- Renewal rate of test solution (frequency/flow rate): ca. 2.7 L/hr (equivalent to 6 volume replacements / day)
- No. of organisms per vessel: 6
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Upper Saginaw Bay of Lake Huron

OTHER TEST CONDITIONS
- Adjustment of pH:
- Photoperiod: 16 hrs light / 8 hrs dark
- Light intensity: 615 - 795 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
survival, fecundyity, fertilization success and clinical signs were observed daily. At test termination (Day 21), secondary sex characterization (body color, pattern, body shape), body weight, length, tubercle score, gonadal staging and histopathology and plasma vitellogenin.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 0.1
- Justification for using less concentrations than requested by guideline: reference study by Cairns and Nebker (see IUCLID Ch. 6.1.2)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.748 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

vitellogenin level

Remarks on result:

other:

Remarks:

females

Effect concentrations 2

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

> 79.8 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality

Effect concentrations 3

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

7.62 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

fecundity

Effect concentrations 4

Key result

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

7.62 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

fertility

Any other information on results incl. tables

Table 1. Adult Fish Survival (21 days)

	Males			Females
Mean-Measured Test Concentration [mg/L]	Number of organisms at the test initiation	Number of organisms at the end oft he test	% Survival	Number of organisms at the test initiation	Number of organisms at the end oft he test	% Survival
Control	8	8	100	16	16	100
0.748	8	8	100	16	16	100
7.62	8	8	100	16	16	100
79.8	8	8	100	16	16	100

 

Table 2 Size of Fish at Test Termination

	Mean Body Weight [g]		Mean Length [mm]
Mean-Measured Test Concentration [mg/L]	Males	Females	Males	Females
Control	3.62	1.79	52.5	43.4
0.748	4.10	1.65	54.3	42.9
7.62	3.79	1.73	54.3	43.8
79.8	2.76	1.35	52.4	42.8

 

Table 3. Fecundity and Fertilization Success.

Mean-Measured Test Concentration [mg/L]	Mean Fecundity *	Mean Fertilization Success [%]**
Control	31.6	98.4
0.748	32.9	97.8
7.62	29.9	98.0
79.8	1.80	29.3

* - Fecundity is calculated as the number of eggs per surviving female per reproductive day per replicate

** - Fertilization success (%) is calculated as the number of embryos divided by the number of eggs, multiplied by 100

Table 4. Nuptial Tubercle Score

Mean-Measured Test Concentration [mg/L]	Mean Tubercle Score Males	Mean Tubercle Score Females
Control	31	0
0.748	32	0
7.62	31	0
79.8	23	0

 

Table 5. Gonado-Somatic Index (GSI)

Mean-Measured Test Concentration [mg/L]	Mean GSI * Males	Difference to Control [%]	Mean GSI* Females	Difference to Control [%]
Control	1.25	0	13.06	0
0.748	1.25	-0.20	13.94	-0.66
7.62	1.30	4.20	14.44	-0.41
79.8	1.20	-4.4	9.36	-70.26

* - Gonado-somatic index (%) is calculated as gonad weight (to the nearest 0.1 mg) / body weight (mg) x 100

Table 6. Gonadal Staging.

Mean-Measured Test Concentration [mg/L]	Median Stage Males	Median Stage Females
Control	2	3
0.748	2	3
7.62	2	3
79.8	2	3

 

Table 7.Plasma Vitellogenin

Mean-Measured Test Concentration [mg/L]	Mean Males [ng/mL plasma]	Difference to Control [%]	Mean Females [mg/mL plasma]	Difference to Control [%]
Control	3480	0	154.25 x 10^6	0
0.748	5080	46.24	147.55 x 10^6	-4.33
7.62	1780	-48.79	86.55 x 10^6	-43.89
79.8	396	-88.63	35.2 x 10^6	-77.18

Applicant's summary and conclusion

Validity criteria fulfilled:

yes