Octanoic acid, mixed tetraesters with 2-ethylhexanoic acid, heptanoic acid and pentaerythritol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Atsugi breeding center
- Age at study initiation: (P) 10 wks
- Weight at study initiation: (P) Males: 330.8-420.4 g; Females: 207.2-245.0 g
- Housing: 2 animals per cage at mating
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0-25.0
- Humidity (%): 40.0-75.0
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Doses were based on a 14 d range-finding study.

DIET PREPARATION
- Rate of preparation of diet (frequency): Prepared up to 8 days before dosing
- Storage temperature of food: Room temperature and shade

VEHICLE
- Justification for use and choice of vehicle (if other than water): For solubility
- Concentration in vehicle: 5, 15, 50% w/v
- Lot/batch no. (if required): V2P1825

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of test substance was analyzed by GC method

Duration of treatment / exposure:

42 days for males and 42-54 days for females

Frequency of treatment:

Daily once

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12 animals (5 animals for satelite groups)

Control animals:

yes, concurrent vehicle

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during breeding period and recovery period, twice daily during dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week

Functional behavior: Day 42 in all groups and Day 14 of recovery for satellite groups

BODY WEIGHT: Yes
- Time schedule for examinations: for males weekly and at necropsy, for females weekly during dosing and on Days 0, 7, 14 and 20 of gestation and on Days 0 and 4 of lactation and at necropsy

FOOD CONSUMPTION: weekly

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- How many animals: 5 animals/sex/dose including the satellite groups

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: No data
- How many animals: 5 animals/sex/dose including the satellite groups

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Fisher’s exact test (significance level: 5%) was performed on the function test results. Among the histopathological findings in the test substance treatment groups, the graded data was tested for significance in relation to the control group using Mann-Whitney U-testing (significance level: 5%), while the total values for positive grades were tested for significance with the control group using Fisher’s exact one-sided testing (significance level: 5%). For the other data, the values obtained for each individual or the mean values for each litter were used as single samples, and these samples were compared with the values within the satellite group or within the other groups. Where two groups were used in the analysis, F-testing was performed first, followed by Student t-testing if no significant differences were observed. If a significant difference was observed in the F-testing, Aspin-Welch testing was performed. When three or more groups were used in the analysis, the Bartlett method was used first in order to test for uniformity of variance in each group (significance level: 5%).If the variance was uniform, one-way analysis of variance (significance level: 5%) was performed. If a significant difference was observed between groups, the Dunnett method was used to perform a multiple comparison (significance level: 5%). On the other hand, if the variance in any of the groups was 0 and if the variance was not uniform, Kruskal-Wallis rank testing (significance level: 5%) was performed. If a significant difference was observed between groups, the Dunnett testing method was used to perform a multiple comparison (significance level: 5%).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

A statistically significant increase in haemoglobin and haematocrit in females at 100 mg/kg bw/day and increased prothrombin time in females at 100 mg/kg bw/day were not considered to be treatment related as no effects at higher doses were noted. No effects were observed at the end of the recovery period.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No effects in males or females in main group; statistically significant increase in urea nitrogen (121% of control) in females at 1000 mg/kg bw/day at the end of the recovery period; no effects in males at the end of the recovery period.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Statistically significant increase in absolute brain weight of females at 300 mg/kg bw/day was not considered to be treatment related as no effect at 1000 mg/kg bw/day was noted. A statistically significant increase of relative liver weight (105% of control) in females at 1000 mg/kg bw/day at the end of recovery, was not considered to be treatment related as it was only seen at the end of recovery, the increase was slight and no histopathological effect was found. No effects in males during main test or at end of recovery were noted.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Testes: focal seminiferous tubule atrophy: 3/12 M at 100 and 1/12 M at 300 mg/kg bw/day; no findings at 1000 mg/kg bw/day and no findings at the end of the recovery period; therefore not treatment related.
Kidney: very slight basophilic tubules in cortex in 2/5 control males and 4/5 males at 1000 mg/kg bw/day and in 1/5 female at 1000 mg/kg bw/day; no findings at end of recovery period.
Ovary: follicle cysts and increased number of follicles in closed state and a corresponding marked reduction in corpus luteum in 1/12 female at 1000 mg/kg bw/day; at end of recovery follicle cysts in 1/5 female at 1000 mg/kg bw/day. Other findings were comparable to the control group in incidence and severeness.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no effects related to test substance treatment.

Key result

Critical effects observed:

no

Conclusions:

Under the study conditions, the systemic NOAEL of the substance in rats was determined to be greater than 1000 mg/kg bw/day.

Executive summary:

A study was conducted to determine the repeated dose toxicity of the substance in rats according to OECD Guideline 422, in compliance with GLP. A combined repeated dose-reproductive developmental screening study was conducted in rats in which the test substance was administered to groups of 12 animals per sex via oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day for 42 days (males) or 42 -54 days (for females). Mortality, clinical signs, food consumption and body weight measurements were performed on a regular basis during the dosing period. Hematological and clinical chemistry examinations were performed before scheduled necropsy. Terminal body weight and organ weights were determined after the necropsy along with gross pathological and histopathological examinations of the major organs. Apart from few non-treatment related effects, no adverse effects were observed in any of the treated groups. Under the study conditions, the systemic NOAEL of the substance in rats was determined to be greater than 1000 mg/kg bw/day (MHLW, 2005).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,2-bis[[(2-ethyl-1-oxohexyl)oxy]methyl]propane-1,3-diyl bis(2-ethylhexanoate)

EC Number:

230-743-8

EC Name:

2,2-bis[[(2-ethyl-1-oxohexyl)oxy]methyl]propane-1,3-diyl bis(2-ethylhexanoate)

Cas Number:

7299-99-2

Molecular formula:

C37H68O8

IUPAC Name:

3-[(2-ethylhexanoyl)oxy]-2,2-bis{[(2-ethylhexanoyl)oxy]methyl}propyl 2-ethylhexanoate

Test material form:

other: Liquid, colorless and transparent

Details on test material:

- Name of test material (as cited in study report): Hexanoic acid, 2-ethyl, 2,2-bis[[(2-ethyl-l-oxohexyl)oxy]methyl]-1,3-propanediyl ester- Molecular formula (if other than submission substance): C37H68O8- Molecular weight (if other than submission substance): 640.94- Physical state: Liquid, colorless and transparent- Analytical purity: 98.4% (GC%)- Stability under test conditions: stable- Storage condition of test material: Room temperature, shade

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CHARLES RIVER LABORATORIES JAPAN, INC. Atsugi breeding center
- Age at study initiation: (P) 10 wks- Weight at study initiation: (P) Males: 330.8-420.4 g; Females: 207.2-245.0 g
- Housing: 2 animals per cage at mating
- Diet: ad libitum
- Water: ad libitum- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0-25.0
- Humidity (%): 40.0-75.0
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hrs. dark/12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on mating procedure:

- M/F ratio per cage: 1/1- Length of cohabitation: up to 2 weeks- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as Day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of test substance was analyzed by GC method (mean content 107-110%)

Duration of treatment / exposure:

42 days for males and 42-54 days for females

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

12 animals (5 animals for satelite groups)

Control animals:

yes, concurrent vehicle

Positive control:

Not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily during breeding period and recovery period, twice daily during dosing

DETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: once a week

BODY WEIGHT: Yes
- Time schedule for examinations: for males weekly and at necropsy, for females weekly during dosing and on Days 0, 7, 14 and 20 of gestation and on Days 0 and 4 of lactation and at necropsy

Oestrous cyclicity (parental animals):

Mean time of estrous cycle and incidence of animals with estrous cycle other than a 4 day cycle per dose was determined beginning at the start of dosing until confirmation of mating.

Sperm parameters (parental animals):

Not applicable

Litter observations:

Number of (live) pups and sex ratio on Days 0 and 4.

Postmortem examinations (parental animals):

SACRIFICE- Male animals: Animals were sacrificed 43 days after satrting dosing or 16 days after starting recovery period. - Maternal animals: Animals were sacrificed 15 days after starting lactation. Animals that did not deliver were saclificed 26 days after pregnancy. For satelite group, sacrifice was done 15 days after starting recovery period.

Postmortem examinations (offspring):

Not applicable

Statistics:

Fisher’s exact test (significance level: 5%) was performed on the function test results, on the incidence of animals showing changes in estrous cycle, on the copulation rate, on the impregnation rate and on the incidence of morphological abnormalities in the offspring.Among the histopathological findings in the test substance treatment groups, the graded data was tested for significance in relation to the control group using Mann-Whitney U-testing (significance level: 5%), while the total values for positive grades were tested for significance with the control group using Fisher’s exact one-sided testing (significance level: 5%).For the other data, the values obtained for each individual or the mean values for each litter were used as single samples, and these samples were compared with the values within the satellite group or within the other groups. Where two groups were used in the analysis, F-testing was performed first, followed by Student t-testing if no significant differences were observed. If a significant difference was observed in the F-testing, Aspin-Welch testing was performed. When three or more groups were used in the analysis, the Bartlett method was used first in order to test for uniformity of variance in each group (significance level: 5%).If the variance was uniform, one-way analysis of variance (significance level: 5%) was performed. If a significant difference was observed between groups, the Dunnett method was used to perform a multiple comparison (significance level: 5%). On the other hand, if the variance in any of the groups was 0 and if the variance was not uniform, Kruskal-Wallis rank testing (significance level: 5%) was performed. If a significant difference was observed between groups, the Dunnett testing method was used to perform a multiple comparison (significance level: 5%).

Reproductive indices:

Copulation index, Fertility index, Gestation index, Imlantation index, Delivery index.

Offspring viability indices:

Birth index, Live birth index, Viability index.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle: no treatment related effect on incidence and length of estrous cycle

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Reproductive performance: No significant differences were observed between the control group and any of the treatment groups in impregnation rate, number of days to copulation or number of estrous during this period. No significant differences were observed for corpus luteum count, implantation count or implantation rate between control and treated groups.

Systemic toxicity details for parental animals are described at 7.5.1 Repeated dose toxicity: oral section.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no effects related to test substance treatment.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

No effect on birth index, live birth index, viability index or sex ratio was observed. Morphological observations on Day 0 of lactation showed a kinked tail in one pup at 1000 mg/kg bw/d. As no tail abnormalities were observed in any of the other animals, this was considered to be a spontaneous malformation. At necropsy no tail abnormalities were found (not clear if pup with kinked tail had died or not).

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no effects related to test substance treatment.

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the NOAEL for fertility and fetal toxicity of the substance in rats was determined to be greater than 1000 mg/kg bw/day.

Executive summary:

A study was conducted to determine the repeated dose toxicity of the substance in rats according to OECD Guideline 422, in compliance with GLP. A combined repeated dose-reproductive developmental screening study was conducted in rats in which the test substance was administered to groups of 12 animals per sex through oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day for 42 days (males) or 42 -54 days (for females). Males and females were co-habited in 1:1 ratio in the cages during the dosing period for a period of 2 weeks till there is proof of pregnancy. Mortality, clinical signs, food consumption and body weight measurements were performed on a regular basis during the dosing period. After the scheduled necropsy of the pregnant females, reproductive and fetal developmental parameters were examined. Apart from few non-treatment related effects, no adverse effects were observed in any of the treated groups. Under the study conditions, the NOAEL for fertility and fetal toxicity of the substance in rats was determined to be greater than 1000 mg/kg bw/day (MHLW, 2005).