1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-rich

Endocrine disrupter testing in aquatic vertebrates – in vivo

Administrative data

Endpoint:

fish life cycle: reproduction, (sub)lethal effects

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The reliability is rated 2 because the study generally followed standard test guidelines, applied valid scientific testing principles, and the results were reviewed for reliability and assessed as valid. However, the study was not conducted under GLP.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

fish life cycle toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

The reliability is rated 2 because the study generally followed standard test guidelines, applied valid scientific testing principles, and the results were reviewed for reliability and assessed as valid. However, the study was not conducted under GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 850.1500 (Fish Life Cycle Toxicity)

Version / remarks:

1996

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test) with evaluation of two generations

Version / remarks:

1992

Deviations:

not applicable

Principles of method if other than guideline:

A specific guideline for a multi-generation test for Medaka was not available at the time of this study. A detailed protocol was developed based on the EPA life cycle test with considerations for the test species as listed in OECD 210. Medaka were used because they are sensitive to estrogen and estrogenic compounds producing ovotestis and other reproductive effects. Medaka reach sexual maturity in 40 to 60 days post-hatch, which allows for a multigeneration study to be completed in one year. The test substance was administered via the diet since this is the major route of exposure to hydrophobic compounds.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

The diets were stored at -20°C ± 2°C for the duration of the study to prevent spoilage. The feed was analyzed by GC-MSD for concentration verification purposes prior to, mid(day 136), and at termination (day 284) of the study.

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

The test substance was administered through the diet.
Diet Preparation:
A single dietary concentration was prepared at a nominal loading of 20 µg/g. The exposure concentration represented a reasonable worst-case scenario based on US EPA field measurements and equilibrium partitioning theory predictions for concentrations in prey organisms.
The experimental diet was prepared by adding the test substance to Tetramin flake fish food (5% lipid content) with the help of acetone as a solvent in order to evenly distribute the test substance in the fish food. The solvent was allowed to evaporate overnight under ambient laboratory conditions. An acetone solvent control was included with an untreated control group and prepared under similar conditions.
Test substance
0.1068g of the test substance was brought up to a final volume of 100 ml with acetone for a final stock concentration of 1068 µg/ml. Twenty milliliters of the stock solution was then added to 1000g of Tetramin. The mixture was mixed for approximately 30 minutes, and then allowed to sit overnight, uncovered, at room temperature to allow the acetone to evaporate.
Feed Control (Solvent Control)
A solvent control feed was prepared by adding 20 ml of acetone to 1000g of Tetramin. The mixture was mixed for approximately 30 minutes, and then allowed to sit overnight, uncovered, at room temperature to allow the acetone to evaporate.
The diets were stored at -20°C ± 2°C for the duration of the study to prevent spoilage.

Test organisms (species):

Oryzias latipes

Details on test organisms:

Husbandry and Acclimation
Cultured in the Environmental Toxicology laboratory. Original stock population was supplied by the Gulf Coast Research Laboratory, Ocean Springs, MS.
Fish were held under flow-through conditions and were observed for parasites and disease prior to use in the test. Fish were acclimated to test conditions prior to the test and were not treated for disease or parasites before use in this study. Eggs/embryos for the study were collected from adults cultured in the laboratory.
Larval fish were fed daily with Artemia (brine shrimp). Juvenile and adult fish were fed daily with Salmon Starter, Tetramin®, and/or Artemia (brine shrimp).

Test type:

flow-through

Water media type:

freshwater

Remarks:

laboratory dilution water was a mixture of carbon filtered well water and water dialyzed by reverse osmosis. The quality was monitored weekly (DO, pH, alkalinity, hardness, conductivity), monthly (TOC), semi-annual (pollutant) and annual (bactericidal).

Limit test:

yes

Total exposure duration:

284 d

Remarks on exposure duration:

Multi-generational test, two full generations. F0: ~14 dph (days post hatch) larval to adults (~140 dph) F1: embryos to adults (~140 dph) F2: embryo to juveniles (~45 dph)

Test temperature:

Temperature was measured daily in each replicate chamber and was maintained at 25°C ± 2°C (per OECD 210 for this species) for the duration of the study.*
Average (± Std dev) test temperature:
Control (untreated, negative): 25.2 ± 0.8 C;
Solvent Control: 25.1 ± 0.9 C;
Test Substance: 25.3 ± 0.9 C

*A few brief excursions (<4% per individual tank) were noted where the temperature fell from 0 to 1.7°C below range. These were not considered to have impacted the integrity of the study.

pH:

Control (untreated, negative): 6.5 - 7.6;
Solvent Control: 6.9 - 7.7;
Test Substance: 6.8 - 7.7

Dissolved oxygen:

Control (untreated, negative): 7.0-8.8 mg/L;
Solvent Control: 7.1-8.7 mg/L;
Test Substance: 7.1-8.8 mg/L

Nominal and measured concentrations:

A single dietary concentration of the test substance was prepared at a nominal loading of 20 µg/g.
Samples were analyzed in triplicate; mean measured concentration of the test substance in the test diet was as follows:
pre-study: 18.35 ± 0.40 µg/g
mid-study (Day 136): 24.5 ± 1.44 µg/g
termination (Day 284): 22.7 ± 081 µg/g.
The overall mean measured exposure concentration of 21.9 µg/g ± 3.08 in the diet (with 5% lipid content) yields an exposure concentration of ~438 ug/g lipid.

Details on test conditions:

Methods:
A flow-through exposure system was constructed to provide a sufficient volume of water to the exposure chambers. Water flow to the test chambers was regulated using a flow-meter and needle valve setup. The flow rate (approximately 250 ml/min) of dilution water into a temperature equilibration tank for each treatment was monitored daily and adjusted as necessary in order to maintain optimal water quality for the test organisms. A splitter box then delivered dilution water to each of five replicate chambers prepared for each of the three treatment groups (treated, solvent control, untreated control).

Test chambers were 19 liter glass aquaria with stainless steel standpipes cut to allow a test volume of 10 liters of water. The flow rate to individual replicate test chambers was ca. 50 ml/min, providing ca. 7 volume replacements daily. Maximum organism loading rate (based on adult body weight at termination) remained below 0.2 g/L/d or an instantaneous loading of 1.2 g/L; consistent with the OECD 240 specification of ≤ 0.5 g/L/d and not exceeding 5 g/L at any time.

Diurnal light: approximately 16 hours light and 8 hours dark, controlled by an automatic timer. Light levels were ca. 55 foot candles, measured at least once per week using a hand-held light meter.

Embryos collected during the study were hatched in 250 ml glass beakers under continuous aeration. Test water in the hatching chambers was replaced as needed to preserve water quality and maintain embryo health.

During overlap between generations, juvenile fish were housed in glass cylinders suspended in the test chambers to prevent losses due to consumption by adult fish. Replicate integrity was maintained throughout the study.

The F0 generation was started with 50 larval Medaka per replicate (a total of 250 fish per group) and were fed the appropriate diet at a rate of 5% body weight per day. The food ration was regularly adjusted to compensate for growth of the fish during the study. Feeding was also supplemented twice weekly with freshly hatched brine shrimp (Artemia sp.), after day 46, due to relatively low growth observed.

Observations for mortality were performed and recorded daily. Dead organisms were recorded and removed from the test chambers. Test chambers were also cleaned periodically to remove accumulated organic material. Once adults reached sexual maturity (about 40 to 60 days post-hatch (DPH)), eggs were collected on sponge filters. Egg production was observed for approximately 3 to 4 weeks and recorded before eggs were collected for hatching of the next generation. Eggs for hatching were collected once all replicates were observed producing eggs. During the embryo-rearing stages of the study the rearing chambers were observed daily and all embryos with fungus or an opaque appearance were removed.

Eggs from each replicate were hatched to provide 50 larval fish for the next generation. At termination (~142 DPH), population, individual, and biochemical test parameters were evaluated. Male and female fish were processed separately. Fish were evaluated for morphometric parameters such as wet weight, total length, gonad weight, and gonadal-somatic index.

Feeding was supplemented three times weekly with freshly hatched brine shrimp in the F1 and F2 generations. As with the original population (F0), the F1 fish were allowed to spawn (F2 generation) and fecundity, egg viability, and embryo development were evaluated. Adult F1 fish were also evaluated based on histopathology, morphological characteristics, and biochemical parameters. F1 larvae were evaluated for lesion occurrence, stage development, post-hatch survival and growth. The F2 larvae were allowed to grow out until 42 DPH, at which time they were weighed, measured, sacrificed, and processed for histopathology evaluation.

Reference substance (positive control):

no

Duration:

284 d

Dose descriptor:

NOEC

Effect conc.:

>= 21.9 other: ug/g feed

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: gonadal histology, larval development, secondary sexual characteristics, histopathology, GSI, HSI, sex ratio, vitellogenin, testosterone

Key result

Duration:

284 d

Dose descriptor:

NOEC

Effect conc.:

>= 21.9 other: ug/g feed

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: larval and adult mortality, growth, fertility

Details on results:

A detail description of the endpoints with supporting tables is provided in the attachment in the attached background material field.
There were no statistically significant effects in the treatment group when compared to the negative or solvent control groups for any of the following population relevant nor mechanistic endpoints:
population relevant:
Survival: larval F1, F2; Juvenile F2; Juvenile-Adult F0, F1†.
Hatch: embryo F1, F2.
Growth - body weight: juvenile F0, F1 (m/f), F2; subadult F0, F1 (m/f); adult F0 (m/f), F1 (m‡/f†).
Growth - total length: juvenile F1 (m/f), F2; subadult F0, F1 (m/f).
Fecundity: eggs/female/day 0 adult F0, F1.
Time to first spawn: subadult F0, F1.
mechanistic:
Sex ratio: adult F0, F1
Histological - organ development / lesion examination of the brain, digestive system, liver, kidney, gonads, and skeleton: F0, F1, F2.
Secondary sex characteristics - phenotypic and histological gender classification for both males and females showed 100% agreement: juvenile F0, F1, F2; subadult F0, F1; adult F0, F1;
no adverse effects on larval male gonad differentiation: F1, F2.
Histopathology – liver: hepatic somatic index (HSI): adult F0 (m/f), F1 (m‡/f).
Histopathology – gonad: gonadal somatic index (GSI): adult F0 (m/f), F1 (m/f).
Biochemical - EROD activity: adult F1 (m/f).
Biochemical - testosterone metabolism: adult F1 (m/f)
Biochemical - hepatic vitellogenin: adult F0 (m/f), F1 (m/f)

†There were slight but statistically significant (Dunnett's, p=0.05) differences between the treatment and the solvent control for juvenile-adult survival (F1), adult female body weight (F1); yet the treatment group was not significantly different from the (negative) control.

‡There were slight but statistically significant (Dunnett's, p=0.05) differences between the solvent control and the (negative) control for adult male body weight (F1), HSI (F1 m); yet the treatment group was not significantly different from either control.

Results with reference substance (positive control):

A positive control was not included.

Reported statistics and error estimates:

Proportion data were arcsine square-root transformed. If data passed normality (Shapiro-Wilks) and homogeneity (Levine), Dunnett's method was used; if not, a nonparamentric comparison with control - Dunn method was used.
An Analysis of Variance (ANOVA) was used to determine differences in the groups based on survival, growth, and fecundity. Embryo developmental stages and hatch were evaluated in a separate peer-reviewed article using a one-way ANOVA using the Sigma Stat program (Patyna P. et. al. (2006). Hazard evaluation of diisononyl phthalate and diisodecyl phthalate in a Japanese medaka multigenerational assay. Ecotoxicol. Environ. Safety 65(1): 36-47).

Validity criteria fulfilled:

yes

Remarks:

The study pre-dated guidelines for multi-generation test on Medaka. Concentration was maintained ± 20% of mean. DO >60% saturation. Temp 25 ± 2C with a few minor excursions. Survival was >80% (larval) and 89 to 94% (juvenile-adult) in the controls.

Conclusions:

Di-isononyl phthalate ester (DINP) did not produce significant effects on the development, growth, survival or reproduction in the Medaka fish (Oryzias latipes) exposed to DINP through the diet at a mean measured concentration of 21.9 ug/g feed. Additionally, there were no significant histological or biochemical effects on testosterone metabolism, EROD activity, or vitellogenin induction.

Executive summary:

Specific guidelines for the study were not available. Therefore, the study was performed following procedures outlined in existing chronic fish toxicity test guidelines. The results showed that dietary exposure (mean measured concentration 21.9 ug/g feed) to di-isononyl phthalate ester (DINP) did not produce significant effects on survival, development, growth and reproduction in Oryzias latipes in a 284 -day multi-generation toxicity study. Phenotypic and histologicial sex determination were in 100% agreement. Based on the lack of VG induction in male groups, DINP was not estrogenic. Taken holistically, the data demonstrate DINP is not estrogenic/antiestrogenic or androgenic/antiandrogenic.

Data source

Reference

Reference Type:

other: Study report; company data

Title:

Unnamed

Year:

2002

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD TG 240 (Medaka Extended One Generation Reproduction Test (MEOGRT))

Principles of method if other than guideline:

A specific guideline for a multi-generation test for Medaka was not available at the time of this study. A detailed protocol was developed based on the EPA life cycle test with considerations for the test species as listed in OECD 210. Medaka were used because they are sensitive to estrogen and estrogenic compounds producing ovotestis and other reproductive effects. Medaka reach sexual maturity in 40 to 60 days post-hatch, which allows for a multigeneration study to be completed in one year. The test substance was administered via the diet since this is the major route of exposure to hydrophobic compounds.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-rich

EC Number:

271-090-9

EC Name:

1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-rich

Cas Number:

68515-48-0

Molecular formula:

C26 H42 O4

IUPAC Name:

1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl esters, C9-rich

Details on test material:

Substance type: commercial product

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The diets were stored at -20°C ± 2°C for the duration of the study to prevent spoilage. The feed was analyzed by GC-MSD for concentration verification purposes prior to, mid(day 136), and at termination (day 284) of the study.

Test solutions

Vehicle:

yes

Remarks:

acetone

Details on test solutions:

The test substance was administered through the diet.
Diet Preparation:
A single dietary concentration was prepared at a nominal loading of 20 µg/g. The exposure concentration represented a reasonable worst-case scenario based on US EPA field measurements and equilibrium partitioning theory predictions for concentrations in prey organisms.
The experimental diet was prepared by adding the test substance to Tetramin flake fish food (5% lipid content) with the help of acetone as a solvent in order to evenly distribute the test substance in the fish food. The solvent was allowed to evaporate overnight under ambient laboratory conditions. An acetone solvent control was included with an untreated control group and prepared under similar conditions.
Test substance
0.1068g of the test substance was brought up to a final volume of 100 ml with acetone for a final stock concentration of 1068 µg/ml. Twenty milliliters of the stock solution was then added to 1000g of Tetramin. The mixture was mixed for approximately 30 minutes, and then allowed to sit overnight, uncovered, at room temperature to allow the acetone to evaporate.
Feed Control (Solvent Control)
A solvent control feed was prepared by adding 20 ml of acetone to 1000g of Tetramin. The mixture was mixed for approximately 30 minutes, and then allowed to sit overnight, uncovered, at room temperature to allow the acetone to evaporate.
The diets were stored at -20°C ± 2°C for the duration of the study to prevent spoilage.

Test organisms

Aquatic vertebrate type:

fish

Test organisms (species):

Oryzias latipes

Details on test organisms:

Husbandry and Acclimation
Cultured in the Environmental Toxicology laboratory. Original stock population was supplied by the Gulf Coast Research Laboratory, Ocean Springs, MS.
Fish were held under flow-through conditions and were observed for parasites and disease prior to use in the test. Fish were acclimated to test conditions prior to the test and were not treated for disease or parasites before use in this study. Eggs/embryos for the study were collected from adults cultured in the laboratory.
Larval fish were fed daily with Artemia (brine shrimp). Juvenile and adult fish were fed daily with Salmon Starter, Tetramin®, and/or Artemia (brine shrimp).

Study design

Test type:

flow-through

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

284 d

Remarks on exposure duration:

Multi-generational test, two full generations. F0: ~14 dph (days post hatch) larval to adults (~140 dph) F1: embryos to adults (~140 dph) F2: embryo to juveniles (~45 dph)

Test conditions

Nominal and measured concentrations:

A single dietary concentration of the test substance was prepared at a nominal loading of 20 µg/g.
Samples were analyzed in triplicate; mean measured concentration of the test substance in the test diet was as follows:
pre-study: 18.35 ± 0.40 µg/g
mid-study (Day 136): 24.5 ± 1.44 µg/g
termination (Day 284): 22.7 ± 081 µg/g.
The overall mean measured exposure concentration of 21.9 µg/g ± 3.08 in the diet (with 5% lipid content) yields an exposure concentration of ~438 ug/g lipid.

Details on test conditions:

please see cross-reference entry under Long-term toxicity to fish

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

284 d

Dose descriptor:

NOEC

Effect conc.:

>= 21.9 other: ug/g feed

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: larval and adult mortality, weight, length, fertility

Effect concentrations 2

Key result

Duration:

284 d

Dose descriptor:

NOEC

Effect conc.:

>= 19.3 other: ug/g feed

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: gonadal histology, larval development, secondary sexual characteristics, histopathology, GSI, HSI, sex ratio, vitellogenin, testosterone

Details on results:

A detail description of the endpoints with supporting tables is provided in the attachment in the attached background material field.
There were no statistically significant effects in the treatment group when compared to the negative or solvent control groups for any of the following population relevant nor mechanistic endpoints:
population relevant:
Survival: larval F1, F2; Juvenile F2; Juvenile-Adult F0, F1†.
Hatch: embryo F1, F2.
Growth - body weight: juvenile F0, F1 (m/f), F2; subadult F0, F1 (m/f); adult F0 (m/f), F1 (m‡/f†).
Growth - total length: juvenile F1 (m/f), F2; subadult F0, F1 (m/f).
Fecundity: eggs/female/day 0 adult F0, F1.
Time to first spawn: subadult F0, F1.
mechanistic:
Sex ratio: adult F0, F1
Histological - organ development / lesion examination of the brain, digestive system, liver, kidney, gonads, and skeleton: F0, F1, F2.
Secondary sex characteristics - phenotypic and histological gender classification for both males and females showed 100% agreement: juvenile F0, F1, F2; subadult F0, F1; adult F0, F1;
no adverse effects on larval male gonad differentiation: F1, F2.
Histopathology – liver: hepatic somatic index (HSI): adult F0 (m/f), F1 (m‡/f).
Histopathology – gonad: gonadal somatic index (GSI): adult F0 (m/f), F1 (m/f).
Biochemical - EROD activity: adult F1 (m/f).
Biochemical - testosterone metabolism: adult F1 (m/f)
Biochemical - hepatic vitellogenin: adult F0 (m/f), F1 (m/f)

†There were slight but statistically significant (Dunnett's, p=0.05) differences between the treatment and the solvent control for juvenile-adult survival (F1), adult female body weight (F1); yet the treatment group was not significantly different from the (negative) control.

‡There were slight but statistically significant (Dunnett's, p=0.05) differences between the solvent control and the (negative) control for adult male body weight (F1), HSI (F1 m); yet the treatment group was not significantly different from either control.

Reported statistics and error estimates:

Proportion data were arcsine square-root transformed. If data passed normality (Shapiro-Wilks) and homogeneity (Levine), Dunnett's method was used; if not, a nonparamentric comparison with control - Dunn method was used.
An Analysis of Variance (ANOVA) was used to determine differences in the groups based on survival, growth, and fecundity. Embryo developmental stages and hatch were evaluated in a separate peer-reviewed article using a one-way ANOVA using the Sigma Stat program (Patyna P. et. al. (2006). Hazard evaluation of diisononyl phthalate and diisodecyl phthalate in a Japanese medaka multigenerational assay. Ecotoxicol. Environ. Safety 65(1): 36-47).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The study pre-dated guidelines for multi-generation test on Medaka. Concentration was maintained ± 20% of mean. DO >60% saturation. Temp 25 ± 2C with a few minor excursions. Survival was >80% (larval) and 89 to 94% (juvenile-adult) in the controls.

Conclusions:

Di-isononyl phthalate ester (DINP) did not produce significant effects on the development, growth, survival or reproduction in the Medaka fish (Oryzias latipes) exposed to DINP through the diet at a mean measured concentration of 21.9 ug/g feed. Additionally, there were no significant histological or biochemical effects on testosterone metabolism, EROD activity, or vitellogenin induction.

Executive summary:

Specific guidelines for the study were not available. Therefore, the study was performed following procedures outlined in existing chronic fish toxicity test guidelines. The results showed that dietary exposure (mean measured concentration 21.9 ug/g feed) to di-isononyl phthalate ester (DINP) did not produce significant effects on survival, development, growth and reproduction in Oryzias latipes in a 284 -day multi-generation toxicity study. Phenotypic and histologicial sex determination were in 100% agreement. Based on the lack of VG induction in male groups, DINP was not estrogenic. Taken holistically, the data demonstrate DINP is not estrogenic/antiestrogenic or androgenic/antiandrogenic.

No endocrine mode of action was detected, nor were any adverse effects at the (sub)population level observed in a two generation study on medaka following dietary exposure to DINP.