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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
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.
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
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
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
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
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
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: 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)
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.
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
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
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.
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
Reproductive effects observed:
not specified

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

Conclusions:
The test material had no effect on reproductive performance and no effect on intrauterine development.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2 due to read-across) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to the endpoint discussion for further details).
The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII - IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Justification for analogue read-across

There are no data on the reproduction toxicity of Tetraesters of pentaerythritol with 2-ethylhexanoic acid, heptanoic acid and nonanoic acid (EC 806-879-4). The assessment was therefore based on studies conducted with analogue substances as part of a read-across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the read-across approach is provided in the technical dossier (see IUCLID Section 13).

Toxicity to reproduction

CAS 7299-99-2

A combined repeated dose toxicity and reproduction/developmental toxicity screening study (according to OECD guideline 422 and in compliance with GLP) was performed with Hexanoic acid, 2-ethyl-, 2,2-bis [ [(2-ethyl-1-oxohexyl)oxy] methyl] -1,3-propanediyl ester (Ohta, 2005). Rats were administered 0, 100, 300 and 1000 mg/kg bw/day of the test substance once daily for 42 days (males) and up to 54 days (females) via gavage. All groups had 12 females, while there were 7 males in the control, low-dose and high-dose groups, and 12 males in the mid-dose group. The application started two weeks before mating on test day one and ended on the day of or one day before sacrifice. Day of sacrifice was on test day 42 for the male rats and on lactation day 4 or shortly thereafter for the female rats. A satellite group of 5 rats/sex/dose with a 14-day recovery period was included for the control and high-dose group. No toxicologically relevant effects were seen on parental viability, clinical signs, body weight (gain), food consumption, haematological parameters, clinical chemistry parameters, neurobehavioural parameters, and during macroscopic and microscopic examinations. Therefore the NOAEL for parental systemic toxicity was ≥ 1000 mg/kg bw/day.

In parental animals, no effects on reproductive function (oestrus cycle) or performance (male and female mating and fertility indices, implantation index, and number of corpora lutea and implantation sites, gestation length) were observed, compared with the control animals. The testis weight, epididymis weight, and histological examination of the testes in males as well as the weight and histological examination of the uterus and ovaries in females did not reveal any substance-related effects in the parental animals. One high-dose male was sterile, which is considered to be an incidental occurrence. Therefore, a NOAEL for parental fertility of ≥ 1000 mg/kg bw/day was derived for male and female rats.

The results of the offspring (viability) parameters pre-implantation loss, pre-birth loss, stillbirths, live births, cumulative pup loss on day 4 post-partum, litter size, sex ratio and pup weight were comparable between control and treatment group. The gross pathological examinations did not reveal any treatment-related effects. Based on the lack of effects, the NOAEL for developmental toxicity/teratogenicity is considered to be ≥ 1000 mg/kg bw/day.

 

90-day oral, dermal and inhalation repeated dose toxicity studies, performed with three source substances, are available (Cruzan, 1988;Dulbey, 1992; Müller, 1998). In these studies no treatment-related effects were observed on the weight and histopathological structure of reproductive organ and tissues.

A waiver for the extended one-generation reproductive toxicity study (EOGRTS) was included. The results of the studies performed on source substances, which cover a wide range of reproductive, fertility and developmental parameters, did not show any potential for toxicity to reproduction (fertility) in rats and a potential for developmental toxicity only at high dose level. It is reasonable to assume for the target substance, that the (lack of) effects noted in the available studies will reflect the results of reproductive parameters assessed specifically in an extended one-generation reproduction toxicity study.Therefore performing an extended one-generation reproduction toxicity study (standard configuration or with additional modules)is not scientifically necessary and, considering concerns regarding the use of vertebrate animals for experimental purposes, unjustified.


Short description of key information:
Oral: OECD 422, rat, NOAEL fertility ≥ 1000 mg/kg bw/day

Justification for selection of Effect on fertility via oral route:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Effects on developmental toxicity

Description of key information
Oral: OECD 414, rat, NOAEL development ≥ 1000 mg/kg bw/day
Oral: OECD 414, rat, NOAEL maternal systemic ≥ 1000 mg/kg bw/day
Dermal: OECD 414, rat, NOAEL maternal local = 200 mg/kg bw/day
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (15 dams per group instead of 20, administration on day 0-19 of gestation, limited details on study design)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
(only 15 presumed pregnant females per group, exposure on day 0-19 of gestation, only 2 dose levels, nonstandard dermal exposure, limited details on exposure)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Kingston, N.Y.
- Age at study initiation: approx. 9 weeks
- Mean weight at study initiation: 248 g
- Diet: Purina Certified Rodent Chow #5002 (Meal), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 40 - 60
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Type of wrap if used: open exposure, no wrap
- Time intervals for shavings or clipplings: no data on frequency; clipped, intact skin
- Site: dorsal
Controls: The rats of the control group were clipped and collared. The intact dorsal skin of each rat was stroked with the tip of a syringe, but no test material was applied.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The amount of the test material applied with a syringe was calculated based on the body weight of the animals and the density of the test substance.

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes. To minimize ingestion of the test material, the rats were fitted with cardboard Elizabethan-style collars.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
gestation days 0 - 19
Frequency of treatment:
daily
Duration of test:
The animals were sacrificed on day 20 of gestation.
Remarks:
Doses / Concentrations:
800 and 2000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
15 presumed-pregnant females
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: based on results of a 13-week dermal study previously conducted with the same material
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: signs of pathosis, abortion, premature delivery, and death

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, 3, 6, 10, 13, 16, and 20 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Time schedule for calculation: days 0-3, 3-6, 6-10, 10-13, 13-16, and 16-20

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The thoracic and abdominal cavities were exposed and all organs were examined grossly for evidence of pathosis.

OTHER:
- Clinical chemistry: alanine aminotransferase (ALT), albumin, albumin/globulin ration, alkaline phosphatase (ALP), aspartate aminotransferase (AST), bilirubin, calcium, chloride, cholesterol, creatinine, globuline, glucose, iron, lactate dehydrogenase (LDH), phosphorus, potassium, sodium, sorbitol dehydrogenase (SDH), total protein, triglycerides, urea nitrogen, and uric acid
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead fetuses: Yes
- Other: The ovaries of non-pregnant females were grossly examined and then discarded.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
Statistics:
- Analysis of variances and group comparison using Fisher's Exact or Dunnett's test (maternal biophase and cesarean section data, and fetal data)
- ANOVA and Fisher's Exact test (fetal skeletal data)
- Fisher's Exact test (fetal visceral data)
- SAS procdures, Student-Newman-Keul's multiple comparison test (clinical chemistry data)
P < 0.05
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: slight local effects

Details on maternal toxic effects:
- General observations: neck lesions, red nasal exudate, and chromodacryorrhea in all groups (considered not to be test substance-related as these signs are common in animals that are collared)
- Local effects: mild dermal irritation including erythema and flaking of the skin in the treatment groups
- Body weight: similar to controls in both treatment groups
- Body weight gain: similar to controls in both treatment groups
- Uterine and net body weights: similar to controls in both treatment groups
- Food consumption: similar to control in both treatment groups; only difference (statistically significant) in high dose group on day 13-16: 31.5 g vs. 29.5 g (corresponding control data)
- Necropsy: no remarkable findings were observed
- Fetal status and uterine position: no parameter evaluated appeared to be adversly affected
- Clinical chemistry: no differences between treated and non-treated rats were observed
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
800 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
Effect level:
< 800 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Fetal body measurements: Mean fetal body weights and crown-rump lengths, parameters of body growth and development were normal compared to controls.
- Fetal examination: No malformations or variations were observeed. Bruises were observed on the skin of 4 fetuses from the control group and 2 fetuses form the 800 mg/kg bw/day group (considered to be incidental). One fetus from one dam exposed to 800 mg/kg bw/day was pale in colour. No other remarkable findings were observed during external examination.
- Malformations (lumbar and sacral vertebrae, tail, sternebrae, and ribs) were observed. The incidence was low and there was no apparent dose-response relationship, these effects were considered to be not treatment-related. Comparable incidences of variant skeletal development were observed in both cotnrol and trated fetuses.
- Visceral examinations: A statistically significant (high dose) increase in the number of fetuses with levocardia (placement of the heart in the extreme left hemithorax) was observed. The response appeared to be dose-related.
Levocardia:
Litters: control: 0 (0%); 800 mg/kg bw/day: 2 (14.3%); 2000 mg/kg bw/day: 7 (50%); 14 litters per group examined
Fetuses: control: 0 (0%); 800 mg/kg bw/day: 3 (3.2%); 2000 mg/kg bw/day: 7 (10.1%); 94, 93, and 99 fetuses examined, respectively
Microphthalmia, anophtalmia and "apparent" hydronephrosis were also observed. Variant visceral development was observed in control and treated fetuses.
Abnormalities:
not specified
Developmental effects observed:
not specified

Levocardia (placement of the heart in the extreme left hemithorax) was the only parameter affected in fetuses of dams treated with the test substance during gestation. In other studies, levocardia was observed in control fetuses, too.

Conclusions:
In a developmental toxicity study, pregnant rats were dermally exposed to the test substance. No adverse effects were observed in any maternal or reproductive parameter nor on external and skeletal development of fetuses. Levocardia (placement of the heart in the extreme left hemithorax) was observed in 3.2% and 10.1% of the fetuses exposed in utero to 800 and 2000 mg/kg bw /day, respectively. Thus, the developmental NOAEL was determined to be < 800 mg/kg bw.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
800 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2 due to read-across) and consistent studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to the endpoint discussion for further details).
The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII - IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) and consistent studies from reference substances with similar structure and intrinsic properties. Read-across is justified based on common precursors and breakdown products of hydrolysis and consistent trends in environmental fate, ecotoxicological and toxicological profile (refer to the endpoint discussion for further details).
The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII - IX, 8.7, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.
Additional information

Justification for analogue read-across

Data on developmental toxicity of Tetraesters of pentaerythritol with 2-ethylhexanoic acid, heptanoic acid and nonanoic acid are not available. The assessment was therefore based on studies conducted with analogue substances as part of a read-across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

Developmental toxicity/teratogenicity

CAS 189200-42-8

The developmental toxicity of Fatty acids C8-10, mixed esters with diPE, isoocatnoic acid, PE and triPE was investigated according to OECD guideline 414 (prenatal developmental toxicity study) and under GLP conditions (Trimmer, 1995). 50 male Sprague-Dawley rats were mated with females to achieve groups of 25 pregnant Sprague-Dawley rats, which then received daily oral gavage doses of the test substance at concentrations of 100, 500 and 1000 mg/kg bw/day during gestation day 6 to 15. On day 21 of gestation the animals were euthanized and examined. One female in the 500 mg/kg bw/day group died on GD 7, most likely due to a dosing error. No toxicologically relevant effects were observed on clinical signs, body weight (gain) and food consumption, and no treatment-related findings were noted during gross necropsy in the P-females. The NOAEL for maternal toxicity was found to be 1000 mg/kg bw/day, the highest dose tested. The number of implantations, number of early resorptions was comparable between the control and treatment groups. Examination of fetus litter size and weights, number of late resorptions, offspring viability (number alive and number dead), sex ratio, grossly visible abnormalities, external, head, soft tissue and skeletal abnormalities showed only incidental malformations. The NOAEL for embryo-/fetotoxicity and teratogenicity in rats for Fatty acids C8-10, mixed esters with diPE, isooctanoic acid, PE and triPE was found to be ≥ 1000 mg/kg bw/day.

CAS 67762-53-2

The potential of Fatty acids, C5-9, tetraesters with pentaerythritol to cause developmental toxicity was assessed in a prenatal developmental toxicity study performed according to a protocol adapted from OECD guideline 414 (Feusten, 1988). 15 pregnant dams/dose were exposed to 800 and 2000 mg/kg bw/day via the dermal route during gestation day 0 to 19. The test substance was applied to the clipped skin and left uncovered. To minimize ingestion of the test substance, the rats were fitted with cardboard Elizabethan-style collars. On day 20 of gestation the animals were euthanized and examined. Neck lesions, red nasal exudate, and chromodacryorrhea was noted in all groups, but considered not to be test substance-related as these clinical signs are common in animals that are collared. No toxicologically relevant effects were observed on body weight (gain), food consumption and clinical chemistry parameters in the P-females. Mild dermal irritation, including erythema and flaking of the skin, was observed at the application site of the treated animals. No treatment-related effects on the dam reproductive parameters (gravid uterus weight, number of corpora lutea, number of implantation sites, pre-implantation loss, early resorptions) were observed. The NOAEL for maternal systemic toxicity was set at ≥ 1000 mg/kg bw/day. There were no significant differences between the control and treatment pups in the developmental parameters (post-implantation loss, body weight, sex ratio, viability). The incidence of malformations and variations in the offspring was comparable between the control and treatment groups. A dose-dependent number of offspring had levocardia, although no heart malformations were observed. In the litters the incidence was 0 (0%), 2 (14.3%) and 7 (50%) in the control, low-dose and high-dose group, respectively (14 litters per group examined). In the foetuses the incidence was 0 (0%), 3 (3.2%) and 7 (10.1%) in the control, low-dose and high-dose group, respectively (94, 93, and 99 fetuses examined, respectively). Levocardia has been observed in vehicle control foetuses (Smith et al. 1988) and in the control foetuses conducted in the test laboratory, meaning there is a natural background incidence. The NOAEL for embryo-/fetotoxicity and teratogenicity in rats was considered to be < 800 mg/kg bw/day and the LOAEL = 800 mg/kg bw/day.

CAS 11138-60-6

The potential of Fatty acids, 8-10 (even numbered), di- and triesters with propylidynetrimethanol to cause developmental toxicity was assessed in a prenatal developmental toxicity study performed according to OECD guideline 414 and under GLP conditions (Azuka and Daston, 2004). 25 pregnant dams/dose were exposed to 200, 600 and 2000 mg/kg bw/day of the test substance via the dermal route during gestation day 6 to 15. The test substance was applied to the clipped skin for 6 hours/day under occlusive conditions. On day 20 of gestation the animals were euthanized and examined. Two animals in the control group and one animal in the high-dose group died within 6 hours after first application. These deaths were not considered to be treatment-related and the animals were replaced. No treatment-related effects were observed on body weight (gain) and food consumption in the P-females. The mid- and high-dose levels caused some local skin irritation at the site of application. One dam of the mid-dose group (1/25) had seven early resorptions. This is considered to be an incidental occurrence as there was no dose-dependency and the number was within the historical control range of the test facility. No treatment-related effects on the dam reproductive parameters (number of corpora lutea, implantation sites, pre-implantation loss, resorptions) or on reproductive tissues and organs were observed. The NOAEL for local effects in considered to be 200 mg/kg bw/day. The maternal NOAEL for systemic effects is set at ≥ 2000 mg/kg bw/day.

There were no significant differences between the control and treatment pups in the developmental parameters (post-implantation loss, body weight, sex ratio, viability). The percentage of fetuses/litter with alteration(s) and the percentage of fetuses with bipartite ossification of the thoracic vertebra were significantly increased in the mid-dose group. Because the incidence was not increased in the high-dose group, and as there was no significant difference between treatment and control groups in the percentage of litter with alterations and percentage of foetuses with alteration, this is considered to be an incidental occurrence. The NOAEL for developmental toxicity and teratogenicity was considered to be ≥ 2000 mg/kg bw/day.

A waiver for the prenatal developmental toxicity study in a second species was included. Information is available from developmental toxicity studies in rats performed with source substances. Considering the results of all the studies and taking into account the fact that for the only observed effect - the increase in levocardia - treatment relation and the toxicological relevance can be questioned it is concluded that the registered substance is considered to have a low potential for developmental toxicityand teratogenicity. Furthermore, the preliminary results of a comparative analysis of data on pharmaceutical compounds suggest that the 2nd species does not add significant information for the assessment of developmental effects. For substances for which there is no convincing data indicating a potential difference in relative species sensitivity between the rat and rabbit, performing a developmental toxicity study in a 2nd species would not be justifiable for animal welfare reasons. Therefore, performing a prenatal developmental toxicity study in a 2nd species is considered not to add new information for hazard assessment and therefore is scientifically and, considering concerns regarding the use of vertebrate animals for experimental purposes, morally unjustified.

 

Overall conclusion for developmental toxicity/teratogenicity

There are no available studies on the developmental toxicity and teratogenicity of Tetraesters of pentaerythritol with 2-ethylhexanoic acid, heptanoic acid and nonanoic acid. Therefore analogue read-across from source substances administered via the oral and dermal route was applied. The dermal prenatal developmental toxicity study performed with Fatty acids, C5-9, tetraesters with pentaerythritol using Sprague-Dawley rats resulted in a NOAEL lower than 800 mg/kg bw/day, since an increase in levocardia was observed in the pups of treated groups, compared with the control. However, levocardia may be observed in both treated and control animals, indicating the effect is not toxicologically relevant. No developmental effects were observed in developmental toxicity studies performed with source substances via the oral route (Fatty acids C8-10, mixed esters with diPE, isoocatnoic acid, PE and triPE (CAS 189200-42-8)) and via the dermal route (Decanoic acid, ester with 2-ethyl-2-(hydroxymethyl)-1,3-propanediol octanoate (CAS 11138-60-6)). Due to the bulkiness and high molecular weight of the target substance, the absorption percentage via oral route is expected to be higher than via the dermal route, indicating the results via the oral route give more accurate indications of the systemic effect. Therefore no hazard was identified for developmental toxicity. The overall NOAEL for developmental toxicity is considered to be ≥ 1000 mg/kg bw/day.


Justification for selection of Effect on developmental toxicity: via oral route:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is the most adequate and reliable study based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Justification for selection of Effect on developmental toxicity: via dermal route:
Hazard assessment is conducted by means of read-across from a structural analogue. The selected study is the most adequate and reliable study performed via the dermal route based on the identified similarities in structure and intrinsic properties between source and target substance and overall assessment of quality, duration and dose descriptor level (refer to the endpoint discussion for further details).

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Tetraesters of pentaerythritol with 2-ethylhexanoic acid, heptanoic acid and nonanoic acid (EC 806-879-4), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis. Based on the analogue read-across approach, the available data on toxicity to reproduction, fertility and development/teratogenicity does not meet the classification criteria according to Regulation (EC) 1272/2008, and is therefore conclusive but not sufficient for classification.

Additional information

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