Reaction mass of 2,2-bis(hydroxymethyl)propane-1,3-diyl bis(2-ethylhexanoate) and 3-[(2-ethylhexanoyl)oxy]-2-{[(2-ethylhexanoyl)oxy]methyl}-2-(hydroxymethyl)propyl 2-ethylhexanoate and 3-[(2-ethylhexanoyl)oxy]-2,2-bis{[(2-ethylhexanoyl)oxy]methyl}propyl 2-ethylhexanoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

22 Apr - 17 Jun 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Pentaerythritol tetra (2-ethylhexanoate). According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

Details on test material:

- Name of test material (as cited in study report): Pentaerythritol tetra (2-ethylhexanoate)
- Physical state: colourless liquid
- Analytical purity: 96.4%
- Lot/batch No.: TOL-887
- Stability under test conditions: verified
- Storage condition of test material: at room temperature in the dark

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: males: 330.8 - 420.4 g (mean 374.0 g), females: 207.2 - 245.0 g (mean 228.3 g)
- Fasting period before study: no fasting period
- Housing: steel cage with wire floor
- Diet: CE-2 from CLEA Japan, Inc., Tokyo, Japan; ad libitum
- Water: tap water; ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.0 - 24.5
- Humidity (%): 49.0 - 67.0
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
50 w/v% solution was prepared by diluting the test substance in vehicle. And this solution was diluted serially with vehicle to prepare 15 and 5 w / v% solution. Gavage solution was prepared within 8 days before administration, since the stability was verified for 8 days. Prepared samples were stored at room temperature in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): corn oil
- Lot/batch no. (if required): V2P1825

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

0.5 mL of each prepared solution was mixed with hexane to be confirmed by GC. The resulting analytical concenrations were 107 - 110% of the nominal concentrations

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Further matings after two unsuccessful attempts: no
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

Males: 42 days
Females: from Day 14 before mating to Day 4 of lactation
Females(satellite): 42 days

Recovary period :
- Males: 14 days
- Females (satellite): 14 days

Frequency of treatment:

once daily

Duration of test:

Males: 42 days
Females: from Day 14 before mating to Day 4 of lactation
Females(satellite): 42 days

Recovary period :
- Males: 14 days
- Females (satellite): 14 days

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

Males; control: 12 (5 for recovery), 100 mg/kg: 12, 300 mg/kg: 12, 1000 mg/kg: 12 (5 for recovery)
Females; control: 12, 100 mg/kg: 12, 300 mg/kg: 12, 1000 mg/kg: 12
Satellite females: control: 5, 1000 mg/kg: 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a preliminary study, 14 days administration in females and males of 100 mg/kg, 300 mg/kg and 1000 mg/kg showed no effect of general condition, body weight gain and organ weights (liver, kidney and spleen).
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Maternal examinations:

Terminal killing:
- Females: Day 5 of lactation
- Females (satellite): Day 15 of recovery

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Males: Day 0, 7, 14, 21, 28, 35 and 42 of treatment
Males in satellite groups: Day 0, 7, 14, 21, 28, 35, 42 of treatment, Day 7 and 14 of recovery period
Females: Day 0, 7, 14, 21, 28, 35, 42 of treatment and once from Day 0 of lactation to Day 4 of lactation
Females in satellite groups: Day 0, 7, 14, 21, 28, 35, 42 of treatment, Day 7 and 14 of recovery period

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: Day 1, 7, 14, 21, 28, 35, 42 of administration and before sacrifice
Males and females in satellite groups: Day 1, 7, 14, 21, 28, 35 , 42 of administration, Day 1, 7, 14 of recovery period and before sacrifice
Females: Day 1, 7, 14, 21 of treatment, Day 0, 7, 14, 20 of pregnancy, Day 0 and 4 of lactation, and before sacrifice

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 26, if pups were not delivered.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

- Terminal killing: 4 days after birth
- External examinations: Yes: all per litter
- Visceral examinations: Yes: all per litter
- Skeletal examinations: No
- Head examinations: No

Statistics:

Fisher test, Mann-Whitney-U-Test, Student's t-test, Aspin-Welch test, Bartlett test, Kruskal-Wallis test, Dunnett-test

Indices:

Reproductive indices: copulation index, fertility index, gestation index, implantation index, number of corpora lutea, number of implantation index
Offspring viability indices: number of newborns, number of dead pups, delivery index, birth index, live birth index, sex ration on day 0 and day 4, viability index

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No mortality was observed in all groups.
An emaciation was observed at day 40 of treatment (Day 1 of lactation) in the female control group, but this was recovered after one day. Loss of the upper incisor was observed at day 25 - 32 in a female of the 300 mg/kg group but this was considered due to physical shock and were not affected by administration of the test substance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males of the 100 mg/kg group showed body weight loss compared to the control group, but this was not statistically significant. There was no difference in female groups and satellite groups compared with control groups.
Females of the satellite 1000 mg/kg group showed significantly more food consumption during days 29 - 30 compared to the control group. However, this change was considered as not compound-related as no difference in other groups was found, both within the treatment period as well as in the recovery period.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Before mating, the estrous cycle of one animal in the 300 mg/kg group was 4/5-day and in one animal of the 100 mg/kg groups the estrous cycle was 5-day during the administration period. However, no significant difference was found.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Regarding coupling, all cases were successful but one animal in 1000 mg/kg was sterile. One dam in the control group was considered to show poor delivering behavior based on the dirty vagina. However, these changes were not compound-related, since no other significant change was found related to gestation rate, paring days until coupling, frequency of estrous, gestation length in days and delivery behaviour.

ORGAN WEIGHTS (PARENTAL ANIMALS)
At the end of dosing period, a significantly increase of absolute weight of brain was found in females (300 mg/kg), but the change of relative weight was not significant. No significant differences in males between treatment group and the control group were observed.
At the end of recovery period, a significantly increase of relative liver weight in females of the 1000 mg/kg group was noted, while no differences in males were found. The change was not considered as compound-related, since no corresponding abnormalities were observed in the livers.

GROSS PATHOLOGY (PARENTAL ANIMALS)
At the end of dosing period, no abnormalities were observed. At the end of recovery period, a tumor in right hind femur was observed in one male of the control group.

HISTOPATHOLOGY (PARENTAL ANIMALS)At the end of dosing period, localized atrophy of the seminiferous tubules were observed in three males of the 100 mg/kg group and in one male of the 300 mg/kg group. Three of these animals had cell debris in lumen in epidedymis. Livers in males of the 1000 mg/kg and control groups exhibited fatty change in hepatocyte and microgranuloma. In all control group animals and three animals of 1000 mg/kg, eosinophilic bodies were found in cortex of the kidney, while slightly basophilic tubules were found in kidney cortex of two animals in the control group and in four animals of the 1000 mg/kg group. Extramedullary hematopoiesis and deposit of brown pigment was found in the spleen of all animals of the control and the 1000 mg/kg group. In addition, focal degeneration/fibrosis in the myocardium, accumulation of focal foam cell in the adveolus and mineralization of the arterial wall in lung, as well as lymphocytes and neutrophil cellular infiltration in prostate were observed both in control groups and 1000 mg/kg groups. However, these change were not significantly different but accidental.
In one female of the 1000 mg/kg group, follicular cyst, increased arterial follicle and decreased corpus luteum was found in ovary at the end of dosing period. In liver, fatty change in periportal hepatocytes and microgranuloma was observed in all animals of control and 1000 mg/kg groups, respectively. Basophilic tubule in the cortex of the kidney was observed in one animal at 1000 mg/ kg and slight mineralization was found in one control animal and one animal dosed with 1000 mg/kg, respectively. In the spleen, extramedullary hematopoiesis and brown deposit were found in all animals of the control and the 1000 mg/kg group. In addition, focal degeneration/fibrosis in myocardium, mineralization on arterial wall of lung and atrophy in thymus were also found in both control and 1000 mg/kg. However, these change were not significantly different but accidental.
At the end of recovery period, intramembranous ossification and periosteum proliferation were found in one male in control. These histopathological findings are due to fracture of the right femur. In one female of 1000 mg/kg, follicular cyst was found.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
A pup with kinked tail was observed at 1000 mg/kg. However, this external change was regarded as incidental since no other significant external difference was found.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Pentaerythritol tetra(2-ethylhexanoate) had no effect on intrauterine development.