Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
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:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
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 by re-analysis
- Storage condition of test material: at room temperature in the dark

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
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. This solution was diluted serially with vehicle to prepare 5 and 15% 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): test substance is insoluble in water, but not in corn oil
- Lot/batch no. (if required): V2P1825
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating, females were individually caged. After day 18 of pregnancy, they were cage in plastic breeding cages with paper and pulp chips (Paperclean, Japan SLC Inc.,) for nesting.
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
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
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
Control: 7 males, 100 mg/kg: 12 males, 300 mg/kg: 12 males, 1000 mg/kg: 7 males;
12 females per dose;
control and 1000 mg/kg for satellite group; 5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose were based on the results of a preliminary test, where groups of male and female rats received doses of 100, 300 and 1000 mg/kg bw, respectively. No effects on general condition, organ weights neither effects on liver, kidney or spleen was observed in any of the treated animals. Based on this, the maximum dose for the study was set to 1000 mg/kg bw, while the medium and low dose were set to 300 and 100 mg/kg bw, respectively.
- Post-exposure recovery period in satellite groups: 14 days

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day during treatment period and once a day during recovery period

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:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 43 (on day after last treatment) for males, Day 15 of recovery period for satellite animals of both sexes, Day 4 of lactation for dams, Day 26 of pregnancy for non delivered dams
- Anesthetic used for blood collection: Yes (sodium pentobarbital)
- Animals fasted: Yes (18 - 24 hours)
- How many animals: 5 in each group
- Parameters checked: erythrocytes count (RBC), hemoglobin, hematocrit, Mean Cell Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Con. (MCHC), leykocyte count (WBC), differential leukocyte count, platelet, Prothrombin time (PT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 43 (on day after last treatment) for males, Day 15 of recovery period for satellite animals of both sex, Day 4 of lactation for dams, Day 26 of pregnancy for non delivered dams
- Anaesthetic used for blood collection: Yes (pentobarbital sodium)
- Animals fasted: Yes (18 - 24 hours)
- Parameters checked: total protein, albumin, A/G, blood urea nitrogen, creatinine, glucose, total cholesterol, triglyceride, Alkaline phosphatase (ALP), Alanine transaminase (ALT (GPT)), Aspartate transaminase (AST (GOT)), γ-GTP, total bilirubin, inorganic phosphorous, calcium, Na, K, Cl

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of dosing period and at the end of recovery period
- Dose groups that were examined: all group
- Battery of functions tested: pupilary reflex, eyelid reflex, visual placing, withdrawal reflex, Payer's reaction, startle reaction, righting reflex
Oestrous cyclicity (parental animals):
Estrous cycles of all female except for satellite groups were observed before and after start of treatment. Vaginal smear samples were everyday prepared and observed until successful mating. Estrus cycles were divided into proestrus, estrus and anestrus, and frequencies of 4-day cycle, 4/5-day cycle and 5-day cycle were calculated.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight
Litter observations:
Termination:
- Offspring: 4 days after birth

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at day 43 (one day after last treatment) and day 15 of recovery
- Maternal animals: All surviving animals at day 5 of lactation
- Females (satellite): All surviving animals at day 15 of recovery

GROSS NECROPSY
- Gross necropsy consisted of brain, the pituitary gland, spinal cord, heart, bronchotracheal, lungs (including bronchi), liver, kidney, thymus, spleen, adrenal, thyroid, parathyroid glands, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, testis, epididymis, ventral prostate, seminal vesicles including coagulation glands, ovaries, uterus, vagina, bladder, lymph nodes under the jaw, mesenteric lymph nodes, sciatic nerve, femur

HISTOPATHOLOGY
Brain, the pituitary gland, spinal cord, heart, lung and bronchus, liver, kidney, thymus, spleen, adrenal gland, thyroid gland, mandibular lymph node, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, testis, epididymis, prostate, seminal vesicles including coagulation glands, ovary, uterus, vagina, mesenteric lymph node, sciatic nerve, urinary bladder, bone marrow of femur

ORGAN WEIGHT
Brain, thymus, heart, liver, kidneys, spleen, adrenal glands, testes, epididymides
Postmortem examinations (offspring):
SACRIFICE
- All pups were subjected to postmortem macroscopic examination

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

Statistics:
Fisher test, Mann-Whitney-U-Test, Student's t-test, Aspin-Welch test, Bartlett test, Kruskal-Wallis test, Dunnett-test
Reproductive indices:
Copulation index, fertility index, gestation index, implatation index, number of corpora lutea, number of implatation 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: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
incidental effects which were not treatment-related (non adverse)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw: reduced body weight in males (non adverse)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw: reduced body weight in males (non adverse)
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
all changes were found distributed over all groups, thus they were regarded as spontanous incidences (non adverse)
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: 1000 mg/kg bw: significantly more food consumption during days 29 - 30 by satellite group (non adverse)

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
one animal in 1000 mg/kg was sterile (non adverse)

Details on results (P0)

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 (P0)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects were observed in males and females of all dose groups

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw: one kinked tail (incidental)
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Number of new borns, number of live newborns, birth index, live birth index, sex ration on Day 0, number of live pups on Day 4, and viability index on Day 4 and sex ratio on Day 4 showed no significant differences in all dose groups compared to the control group.

CLINICAL SIGNS (OFFSPRING)
A pup with kinked tail was observed in 1000 mg/kg. However, this external change was regarded as incidental, since no other significant external difference was found.

BODY WEIGHT (OFFSPRING)
No difference was found between treatment groups and control group.

GROSS PATHOLOGY (OFFSPRING)
No difference was found between treatment groups and control group.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects were observed in pups of all dose groups

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Estrous cycle of dams:

Dose (mg/kg bw)

0*

100

300

1000

Number of dams examined

12

12

12

12

Pre-treatment period

Number of animals showing type of cycle

 

 

 

 

4-day cycle

12

12

12

12

Treatment period

Number of animals showing type of cycle

 

 

 

 

4-day cycle

12

11

11

12

4/5-day cycle

0

0

1

0

5-day cycle

0

1

0

0

Frequency of animals of which type of estrous cycle was changed after treatment

0/12

1/12

1/12

0/12

Mean times of estrous cycle during mating period (mean ± SD)

1 ± 0.0

1.1 ± 0.3

1.0 ± 0.0

1.0 ± 0.0

*vehicle control

Reproductive performance:

Dose (mg/kg bw)

0*

100

300

1000

Number of pairs examined

12

12

12

12

Number of pair copulated

12

12

12

12

Copulation index (%)

100

100

100

100

Number of pregnant females

12

12

12

11

Fertility index (%)

100

100

100

91.7

Pairing days until copulation (mean ± SD)

2.1 ± 1.0

2.9 ± 1.6

 2.8 ± 1.1

3.4 ± 3.1

*vehicle control

Development of pups up to 4 days of lactation:

Dose (mg/kg bw)

0*

100

300

1000

Number of pregnant females

12

12

12

11

Number of pregnant females with live newborns

12

12

12

11

Gestation index (%)

100

100

100

100

Gestation length in days

22.5 ± 0.5

22.5 ± 0.5

22.3 ± 0.5

22.5 ± 0.5

Number of corpora lutea

16.4 ± 2.5

15.5 ± 1.7

15.8 ± 1.8

16.2 ± 1.0

Number if implantations

14.4 ± 2.8

15.3 ± 1.5

15.3 ± 1.7

15.5 ± 1.0

Implantation index

88.8 ± 18.7

98.5 ± 3.5

97.0 ± 4.7

95.6 ± 5.2

Day 0 of lactation (at birth)

Number of newborns

13.8 ± 2.7

13.5 ± 2.4

14.3 ± 2.1

14.4 ± 1.3

Delivery index

96.2 ± 5.0

88.4 ± 13.2

93.2 ± 6.8

93.0 ± 6.8

Number of live newborns

13.6 ± 2.8

13.2 ± 2.4

14.2 ± 2.2

14.0 ± 1.6

Males

6.5 ± 2.7

7.3 ± 2.5

7.0 ± 2.5

6.4 ± 1.8

Females

7.1 ± 1.8

5.9 ± 1.9

7.2 ± 1.6

7.6 ± 2.5

Birth index (%)

94.6 ± 8.1

86.3 ± 14.2

92.1 ± 6.7

90.5 ± 7.7

Live birth index (%)

98.2 ± 4.5

97.7 ± 6.3

98.9 ± 3.8

97.3 ± 5.0

Sex ratio on day 0

45.5 ± 18.2

53.9 ± 15.9

48.6 ± 12.5

46.0 ± 14.0

Day 4 of lactation

Number of live pups

13.4 ± 3.0

13.1 ± 2.5

14.1 ± 2.2

13.7 ± 1.6

Males

6.4 ± 2.8

7.2 ± 2.4

7.0 ± 2.5

6.3 ± 1.8

Females

7.0 ± 1.9

5.9 ± 1.9

7.1 ± 1.6

7.5 ± 2.4

Viability index

98.6 ± 4.8

99.4 ± 2.2

99.4 ± 2.2

98.2 ± 4.5

Sex ratio on day 4

45.4 ± 18.3

53.8 ± 15.6

48.8 ± 12.3

46.2 ± 14.1

*vehicle control

Mean body weights of pups up to day 4 of lactation (g):

Dose (mg/kg bw)

0*

100

300

1000

Day 0 of lactation (at birth)

Number of live newborns

 

 

 

 

Male

6.5 ± 2.7

7.3 ± 2.5

7.0 ± 2.5

6.4 ± 1.8

Female

7.1 ± 1.8

5.9 ± 1.9

7.2 ± 1.6

7.6 ± 2.5

Mean body weight (g)

 

 

 

 

Male

6.6 ± 0.5

7.1 ± 0.8

6.7 ± 0.9

6.7 ± 0.6

Female

6.4 ± 0.6

6.7 ± 0.8

6.3 ± 0.8

6.3 ± 0.5

Day 4 of lactation (at birth)

Number of live newborns

 

 

 

 

Male

6.4 ± 2.8

7.2 ± 2.4

7.0 ± 2.5

6.3 ± 1.8

Female

7.0 ± 1.9

5.9 ± 1.9

7.1 ± 1.6

7.5 ± 2.4

Mean body weight (g)

 

 

 

 

Male

10.6 ± 0.8

11.7 ± 2.0

10.6 ± 1.6

10.5 ± 1.2

Female

10.7 ± 1.4

11.2 ± 1.9

10.1 ± 1.6

10.0 ± 1.1

*vehicle control

Morphological observations of pups:

Dose (mg/kg bw)

0*

100

300

1000

Dead pups

Number of dead pups

5

5

3

7

Number of missing pups

0

0

1

3

Number of dead pups examined

5

5

2

4

Number of dead pups with external changes

0

0

0

0

Number of dead pups with visceral changes

0

0

0

0

Live pups

Number of newborns examined (at birth)

163

158

170

154

Number of newborns with external changes

0

0

0

1

Kinked tail

0

0

0

1

Number of dead pups examined at day 4 of lactation

161

157

169

151

Number of dead pups with external changes

0

0

0

0

Number of dead pups with visceral changes

0

0

0

0

*vehicle control

Applicant's summary and conclusion

Conclusions:
The test material had no effect on reproductive performance and no effect on intrauterine development.