Reaction product of 3,5,5-trimethyl-hexanoic acid and 2-methylpropanoic acid and pentaerythritol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL (systemic toxicity) =  100 mg/kg bw/day; OECD 421; Armour G. (2012)

NOAEL (Reproduction) =  1000 mg/kg bw/day; OECD 421; Armour G. (2012)

NOAEL (F0 and F1) =  300 mg/kg bw/day; OECD 416; Armour G. (2013)

NOAEL (F2) =  100 mg/kg bw/day; OECD 416; Armour G. (2013)

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Sep 2011 - 25 Apr 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

1995

Deviations:

yes

Remarks:

The deviations were considered to have not affected the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Source and lot/batch No.of test material: JA01YX10
- Expiration date of the lot/batch: Jan 2013
- Purity test date: 95.8 %



RADIOLABELLING INFORMATION (if applicable): N/A




STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL

- Storage condition of test material:bRoom temperature (ca 20 °C), in the dark under Nitrogen
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: stable in the liquid matrix at concentrations of 2 and 200 mg/mL for 24 hours at ambient temperature and for up to 15 days when refrigerated (nominally 2 to 8 ºC). 

- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A



TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Treatment of test material prior to testing: The test material was used as supplied. The test item was prepared for administration as a series of graded concentrations in the vehicle - corn oil.
- Preliminary purification step (if any): Dose range finder was conducted prior to the main experiment ignorer to determine suitable dose levels.

- Final dilution of a dissolved solid, stock liquid or gel: 5 mL/kg
- Final preparation of a solid: N/A



FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A

Species:

rat

Strain:

CD-1

Remarks:

Crl:CD(SD) rats

Details on species / strain selection:

Crl:CD(SD) rats from Charles River (UK) Ltd.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes

- Age at study initiation: 70 days

- Weight at study initiation: Weight range of 332 to 382 g for males and 238 to 270 g for females.
- Fasting period before study: Not Stated

- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.



DETAILS OF FOOD AND WATER QUALITY:

- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet.
- Water (e.g. ad libitum): Polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS

- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.

- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

IN-LIFE DATES: From: To: 28 Sep 2011 - 11 Nov 2011

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
 


DIET PREPARATION

- Rate of preparation of diet (frequency): N/A

- Mixing appropriate amounts with (Type of food): N/A

- Storage temperature of food: N/A
 


VEHICLE

- Justification for use and choice of vehicle (if other than water): The test item was insoluble in water hence corn oil was used

- Concentration in vehicle: 2 mg/mL and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bodyweight,
- Lot/batch no. (if required): Not stated

- Purity: Not stated

Details on mating procedure:

Following a minimum of two weeks of treatment males and females were paired on a one-to- one basis from within the same treatment group for a period of up to two weeks.

Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa and the stage of the oestrous cycle. The day on which evidence of mating was found was designated Day 0 of gestation. Once mating occurred, the males and females were separated and smearing was discontinued.

The pre-coital interval was calculated for each female as the time elapsing between initial pairing and detection of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Detailed records of compound usage were maintained. The amount of test material necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Homogeneity and stability of the test material in the vehicle was performed as part of the 13 week study (Huntingdon Life Sciences Study Number: OWH0019). The homogeneity and stability were confirmed following storage at ambient temperature (nominally 21 °C) for 0, 2 and 4 hours, and refrigerated (nominally 2-8 °C) for 1 day, 8 days and 15 days.

Samples of each formulation prepared for administration in first and last formulation occasions were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL) from all groups. Two samples from each group were analysed and the remainder were retained as contingency.

Duration of treatment / exposure:

15 days before pairing until Day 6 after the birth of the F1 generation.

Frequency of treatment:

Daily

Details on study schedule:

Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed.

F0 females were not dosed if parturition was in progress at the scheduled time of administration.

Animals received the test material or vehicle control formulations orally at a volume-dosage of 5 mL/kg bodyweight, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach.

All animals were dosed in sequence of cage-number within each group, once each day at approximately the same time each day, seven days per week. The volume administered to each animal was calculated from the most recently recorded scheduled bodyweight.

A daily record of the weight of each formulation dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 Control (Corn oil)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 (test item)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3 (test item)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4 (test item)

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

Dose selection rationale: Dose selection was based on the result obtained from a 14 day dose range finder study.

- Rationale for animal assignment (if not random): Animal were assigned randomly

- Fasting period before blood sampling for clinical biochemistry: Not stated

- Rationale for selecting satellite groups: Not included

- Post-exposure recovery period in satellite groups: N/A

- Section schedule rationale (if not random): N/A

- Other: Not stated

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

- Cage side observations checked

DETAILED CLINICAL OBSERVATIONS: Yes

- Time schedule: Twice daily


BODY WEIGHT: Yes 

- Time schedule for examinations: The weight of each adult was recorded during acclimatisation, on the day that treatment commenced (Week 0), weekly thereafter and before necropsy. The weight of each F0 female was also recorded on Days 0, 3, 7, 10, 14, 17 and 20 after mating and on Days 1, 4 and 7 of lactation.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: N/A
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes; The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded on a weekly basis from the start of treatment for the F0 adults until the animals were paired for mating. From these records the mean weekly consumption per animal (g/animal/week) was calculated for each cage. For each F0 female, the weight of food supplied, that remaining and an estimate of any spilled was also recorded for the periods Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and Days 1-3 and 4-6 of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal.


FOOD EFFICIENCY:

- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes



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


OTHER: Not Specified


Oestrous cyclicity (parental animals):

After pairing until mating, smearing was performed using a pipette lavage.

Sperm parameters (parental animals):

Not specified

Litter observations:

All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter. The records maintained were as follows:
Clinical signs: Daily records were maintained for evidence of ill-health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-7 of age.
Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 and 7 of age.
Bodyweight: Individual offspring bodyweights were recorded on Days 1, 4 and 7 of age.

Postmortem examinations (parental animals):

SACRIFICE: Yes

F0 males were killed after Day 7 of lactation of the females.
F0 females surviving until the end of the scheduled study period were killed on Day 7 of lactation. Group 2 female number 58 failed to produce a viable litter so was killed on Day 25 after mating. Two females and their litters (Group 3 number 67 and Group 4 number 71) were killed on Day 21 and 23 after mating respectively as the dam had poor survival prognosis. A further female and litter (Group 3 number 66) was killed on Day 2 of lactation.

GROSS NECROPSY: Yes

All tissues preserved for examination (as specified above) were examined for all animals of Groups 1 (Control) and 4 (1000 mg/kg/day) sacrificed on completion of the scheduled treatment period and was extended to cover the ovaries of females in Groups 2 and 3 (100 and 300 mg/kg/day).

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes

Epididymides, ovaries and testes were subject to histological processing. Those tissues subject to histological processing included the following regions:
-Epididymides (caput, corpus and cauda)
-Ovaries (qualitative evaluation of one section from each ovary)
-Uterus (uterine body with cervix section and oviducts)

Postmortem examinations (offspring):

SACRIFICE:Yes

Offspring were killed on Day 7 of age.

GROSS NECROPSY: Yes
For offspring surviving to scheduled termination, a careful external examination was performed for gross abnormalities and externally normal offspring were discarded without internal examination. Externally abnormal offspring were internally examined and any abnormal tissues were retained in an appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGHTS: Yes

Statistics:

The following sequence of statistical tests was used for bodyweight, food consumption, organ weights and litter data: Bartlett's test, Dunnett's test, Shirley's test and Williams’ test

For litter data, if 75 % of the data (across all groups) were the same value, for example c, Fisher’s Exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate (Angervall and Carlstrom, 1963). The treatment comparisons were made on adjusted group means in order to allow for differences in bodyweight which might influence the organ weights.

Sex ratio were analysed by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz et al 1991). Each treated group was compared to control using a Wald chi-square test. The numerator was Number of males, the denominator was Number of live fetuses.

For gestation length, an exact two-tailed Linear-by-linear test (Cytel 1995), with equally spaced scores, was applied to all groups. If the test was statistically significant (p<0.05), the highest dose group was excluded and the test re-applied. This ‘step-down’ process was repeated until the test was no longer statistically significant (p≥0.05). If the exact version of the Linear-by-linear test could not be calculated (due to the size of the table containing the data), then the asymptotic version was used instead.

Reproductive indices:

Percentage mating = Number animals mating x 100 Animals paired
Conception rate (%) = Number animals achieving pregnancy x 100 Animals mated
Fertility index (%) = Number animals achieving pregnancy x 100 Animals pairing

Offspring viability indices:

The following were calculated for each litter:
Post-implantation survival index (%) = Total number of offspring born x100 Total number of uterine implantation sites
Post-implantation survival index was expressed as 100 % where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = Number of live offspring on Day 1 after littering Total number of offspring born
Viability index (%) = Number of live offspring on Day 7 Number live offspring on Day 1 after littering
Group mean values were calculated from individual litter values.
Percentage males = Number of males in litter x 100 Total number of offspring in litter

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Underactivity, irregular breathing, dark discharge from the vagina, partially closed eyelids and skin pallor observed in three female at 300 and 1000 mg/kg/day

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were three deaths, all in females at the end of gestation/early lactation:
- Two females in the 300 mg/kg/day group (3F 66 &3F 67) with clinical signs of underactivity, irregular breathing, dark discharge from the vagina, partially closed eyelids and skin pallor. Macroscopic examination at necropsy revealed reduced caecum contents, dark fluid in the caecum and jejunum, inactive mammary tissue, a small spleen, firm, dark material in the stomach and dark fluid in the vagina.
- One female in the 1000 mg/kg/day group (4F 71) showing clinical signs of underactivity, fast breathing, and piloerection. Macroscopic examination at necropsy revealed reduced caecum, duodenum, ileum and jejunum contents, pale and inactive mammary tissue, a small spleen, and a stomach distended with food, placental and fetal material. hree male pups and four female pups were found dead before the adult female was killed for welfare reasons, and three live male pups, one live female pup and one dead female pup were present in the cage with this female when she was killed for welfare reasons.

Body weight and weight changes:

not specified

Description (incidence and severity):

There were no clear adverse effects of treatment of upon bodyweights of males or of females before pairing, with treated animals gaining similar amounts of or slightly more weight than controls.
During gestation there appeared to be a slight reduction in weight gain for females at 1000 mg/kg/day, as a consequence of reduced gain in the later period of measurement (Day 17 -20). Bodyweight at Day 1 post partum was slightly low (95 % of control) but gains were similar in all groups to Day 7 post partum when the study ended.

Food consumption and compound intake (if feeding study):

not specified

Description (incidence and severity):

There were no clear effects on food consumption in the weeks before pairing or, for females, during gestation and lactation at dose levels of up to 1000 mg/kg/day.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Description (incidence and severity):

The pre-coital interval was unaffected by treatment, with all animals mating at the earliest opportunity, when the female came into oestrus.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance and fertility as assessed by percentage mating, conception rate and fertility index, were unaffected by treatment. One pairing in the 100 mg/kg/day group was infertile, and could probably be attributed to the male which had small reproductive organs. The isolated nature of this finding suggests that it was unrelated to treatment. With regard to the two females that were killed for welfare reasons around the time of parturition, no live pups were seen for Group 3 female number 67 and was considered to have no live litter because the pups had died before or during parturition. Group 4 female number 71 did have a live litter because there were 4 live pups (as well as 8 dead ones).

The pre-coital interval was unaffected by treatment, with all animals mating at the earliest opportunity, when the female came into oestrus.

The gestation length was within the normal range of 22-23 days, and there was no evidence for a treatment-related shift in the distribution of gestation lengths, although the control group contained a slightly higher proportion of females with a 23 day gestation length. The gestation index was unaffected by treatment.

Reproductive performance in terms of mating, fertility, litter size, growth and survival of the offspring was unaffected by parental treatment with the test item. The microscopic examination of testis, epididymides and ovaries from the F0 generation animals revealed the presence of findings only in the female ovaries, and these changes were considered to be of unclear relationship to treatment with the test item.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the effects seen in utero growth of the offspringon birth weight at 300 and 1000 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adversed effects observed

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Offspring clinical signs were limited to a few instances of cold to touch and bruising. Dark abdomens were observed amongst the offspring of one litter in Group 3 (Litter 69) from Days 2 – 7 of age, and amongst the offspring of two ltters in Group 4 (Litters 74 and 78) on Day 4 or Days 4-5 of age, respectively.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The mean number of implantations, the live litter size on Day 1 and sex ratio showed no adverse effect of parental treatment. The offspring survival up to Day 7 of age was slightly low for some litters in the treated groups but it was considered that there was no dosage related effect.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Offspring bodyweight on Day 1 of age was slightly low (82 % of control) for litters in the highest dose group (1000 mg/kg/day), but by Day 7 of age there was an improvement in relative offspring weight (88/90 % of male/female weight. Birth weight at 300 mg/kg/day was also slightly low (86-88 % of control).

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination of offspring which died or were killed for welfare reasons generally confirmed the last in-life finding of no milk present in stomach. No other macroscopic abnormalities were found amongst these animals.
Macroscopic examination of offspring killed at scheduled termination on Day 7 of age revealed no abnormality.

Histopathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring that died prior to scheduled termination and at scheduled termination on Day 7 of age did not reveal any findings that could be attributed to parental treatment.

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

other: Overal NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: birth weight at 300 and 1000 mg/kg/day.

Key result

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 noted

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Tables containing raw data are attached in full study report.

Conclusions:

Reproductive performance in terms of mating, fertility, litter size, growth and survival of the offspring was unaffected by parental treatment with the test item. Three females were killed for welfare reasons. One at 300 and one at 1000 mg/kg/day were killed during parturition and a second female at 300 mg/kg/day was killed on Day 2 of lactation. Necropsy did not reveal test item related effects, it was concluded that the lower offspring bodyweight and lower maternal weight gain during late lactation seen at these doses and an association with treatment cannot be excluded. The birth weights of litters at 100 mg/kg/day were within the normal control range.

Therefore it was considered that treatment at 300 and 1000 mg/kg/day had biologically significant effects upon the in utero growth of the offspring, but there was no evidence of developmental abnormality. There was no evidence of an effect of treatment upon reproductive performance of the parental generation or that the in utero effect would lead to an effect upon reproductive performance of the F1 generation.

It is concluded that that the No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/day for reproductive performance and 100 mg/kg/day was considered to be the overall NOAEL because of the effects seen on birth weight at 300 and 1000 mg/kg/day.

Executive summary:

OECD 421 (2012) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 421), Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid was administered to 10 male and 10 female Crl:CD(SD) strain rats in corn oil via oral gavage at dose of 100, 300 and 1000 mg/kg/day for up to 5 weeks. In addition, 10 male and 10 female control rats were administered corn oil only.

 

A summary of adult responses to the test item are described below;

Systemic toxicity (F0)

 

Mortality- There were three deaths, all in females at the end of gestation/early lactation. ne at 300 and one at 1000 mg/kg/day were killed during parturition and a second female at 300 mg/kg/day was killed on Day 2 of lactation. Necropsy did not reveal test item related effects, it was concluded that the lower offspring bodyweight and lower maternal weight gain during late lactation seen at these doses and an association with treatment cannot be excluded.  The birth weights of litters at 100 mg/kg/day were within the normal control range.

 

Clinical signs- Underactivity, irregular breathing, dark discharge from the vagina, partially closed eyelids and skin pallor observed in three female at 300 and 1000 mg/kg/day.

Bodyweight- There were no clear adverse effects of treatment of upon bodyweights of males or of females before pairing, with treated animals gaining similar amounts of or slightly more weight than controls. During gestation there appeared to be a slight reduction in weight gain for females at 1000 mg/kg/day, as a consequence of reduced gain in the later period of measurement (Day 17 -20). Bodyweight at Day 1 post partum was slightly low (95% of control) but gains were similar in all groups to Day 7 post partum when the study ended.

  

Food consumption and efficiency-There were no clear effects on food consumption in the weeks before pairing or, for females, during gestation and lactation at doses up to 1000 mg/kg/day.

  

Histopathological changes and Necropsy-No toxicologically significant effects were detected in terminal kill animals of either sex treated with the test item.  However, Changes of an uncertain relationship to treatment were seen in the ovary of females given the test item at 100, 300 and 1000 mg/kg/day. Prominent corpora lutea of pregnancy in the ovaries were more frequently seen in all treated groups than in controls, particularly in females dosed at 1000 mg/kg/day.

  

Organ weights-Organ weights were not affected by treatment. The testes and epididymides of one male (Group 2 M18) were atypically low, but this was considered unlikely to relate to treatment.

 

Reproductive performance (F0)

Mating- No treatment-related effects were detected in mating performance.

Estrous cycle- There was no effect of treatment on estrous cycle regularity during the course of the 2-week pre-pairing assessment period for the Reproductive phase females.  

 

Fertility- The pre-coital interval was unaffected by treatment, with all animals mating at the earliest opportunity, when the female came into oestrus.

 

Gestation length- There was no effect of treatment on gestation length, with all gestation lengths within the expected range of 22 to 23 days.  There was no evidence for a treatment-related shift in the distribution of gestation lengths, although the control group contained a slightly higher proportion of females with a 23 day gestation length. The gestation index was unaffected by treatment.

        

Litter size, viability- The mean number of implantations, the live litter size on Day 1 and sex ratio showed no adverse effect of parental treatment. The offspring survival up to Day 7 of age was slightly low for some litters in the treated groups but it was considered that there was no dosage related effect.

Offspring bodyweight on Day 1 of age was slightly low (82 % of control) for litters in the highest dose group (1000 mg/kg/day), but by Day 7 of age there was an improvement in relative offspring weight (88/90 % of male/female weight. Birth weight at 300 mg/kg/day was also slightly low (86-88 % of control).

 

Therefore, it was considered that treatment at 300 and 1000 mg/kg/day had biologically significant effects upon the in utero growth of the offspring, but there was no evidence of developmental abnormality. There was no evidence of an effect of treatment upon reproductive performance of the parental generation or that the in utero effect would lead to an effect upon reproductive performance of the F1 generation. 

It is concluded that that the No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/day for reproductive performance and 100 mg/kg/day was considered to be the overall NOAEL because of the effects seen on birth weight at 300 and 1000 mg/kg/day. 

This combined toxicity study with reproduction screening in the rat is acceptable and satisfies the guideline requirements for an OECD 421 in the rat.

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 May 2012 - 23 May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted in accordance with national standard and under GLP condition

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

2001

Deviations:

yes

Remarks:

The deviations were considered to have not affected the integrity of the study.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Version / remarks:

Agust 1998

Deviations:

yes

Remarks:

The deviations were considered to have not affected the integrity of the study.

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No 8147, Agricultural Production Bureau.

Version / remarks:

November 24, 2000.

Deviations:

yes

Remarks:

The deviations were considered to have not affected the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Source and lot/batch No.of test material: JA01YX10
- Expiration date of the lot/batch: Jan 2013
- Purity test date: 95.8 %



RADIOLABELLING INFORMATION (if applicable): N/A




STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL

- Storage condition of test material: Room temperature (ca 20 °C), in the dark under nitrogen
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: stable in the liquid matrix at concentrations of 2 and 200 mg/mL for 24 hours at ambient temperature and for up to 15 days when refrigerated (nominally 2 to 8C).

- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A



TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Treatment of test material prior to testing: The test material was used as supplied. The test item was prepared for administration as a series of graded concentrations in the vehicle - corn oil.
- Preliminary purification step (if any): Dose range finder was conducted prior to the main experiment ignorer to determine suitable dose levels.

- Final dilution of a dissolved solid, stock liquid or gel: 5 mL/kg
- Final preparation of a solid: N/A



FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A


Species:

rat

Strain:

Crj: CD(SD)

Remarks:

Crl:CD(SD) rats

Details on species / strain selection:

Crl:CD(SD) rats from Charles River (UK) Ltd.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes

- Age at study initiation: 22-28 days
- Weight at study initiation: Weight range of 135 g to 194 g for males and 108 g to 164 g for females.
- Fasting period before study: Not Stated

- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.



DETAILS OF FOOD AND WATER QUALITY:

- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet.
- Water (e.g. ad libitum): Polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS

- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.

- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

IN-LIFE DATES: From: To: 02 May 2012 - 23 Jan 2013

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Administration details:
-Route : Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
-Treated at: Constant doses in mg/kg/day.
-Volume dose: 5 mL/kg bodyweight.
-Individual dose volume: Calculated from the most recently recorded scheduled bodyweight.
-Control (Group 1): Vehicle at the same volume dose as treated groups.
-Frequency: Once daily at approximately the same time each day.
-Formulation: A daily record of the usage of formulation was maintained based on weights before and after dosing. The difference between actual and expected usage was monitored as a check of correct administration.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Details on mating procedure:

Mating procedure:
- F0 pairing commenced: After 10 weeks of treatment.
- F1 pairing commenced 10 weeks after formal commencement (nominal Day 28 of age).
- Male/female ratio: 1:1 from within the same treatment groups (sibling pairing was not permitted).
- Duration of pairing: Up to 2 weeks.
- Daily checks for evidence of mating: Presence of ejected copulation plugs.
Vaginal smear - examined for the presence of spermatozoa and the stage of the oestrous cycle.
- Day 0 of gestation: When positive evidence of mating was detected.
- Male/female separation: Day when mating evidence was detected.
- Pre-coital interval:Calculated for each female as the time between first pairing and evidence of mating.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Approximately 50 % of the final volume of vehicle was added to the test item and magnetically stirred until the test material had dispersed and homogenous. Specimen formulations were prepared at concentrations of 2 mg/mL and 200 mg/mL .

The stability was assessed following storage at ambient temperature (nominally 21°C) for 0, 2 and 4 hours and 1 day, and refrigeration (nominally 2 to 8 ºC) for 1 day, 8 days and 15 days.

Single samples were taken for assay from the top, middle and bottom of the magnetically stirred formulation and homogeneity was determined by analysis of these samples. Stability was determined from the mean concentration of the test item in the vehicle at each sampling point.

The results of the analysis confirmed that the test item produced an homogenous suspension and was stable in the liquid matrix at concentrations of 2 and 200 mg/mL for 24 hours at ambient temperature and for up to 15 days when refrigerated (nominally 2 to 8 ºC).

Duration of treatment / exposure:

Duration of treatment:
- F0 animals: For 10 weeks before pairing until termination after litters were weaned.
- F1 animals: From weaning for minimum of 10 weeks before pairing and until termination after litters were weaned. Direct treatment of selected F1 offspring commenced at weaning (Day 21 of age). The unselected F1 offspring, and the F2 offspring received no direct treatment but may have been exposed in utero or via the milk.

Frequency of treatment:

Once daily

Details on study schedule:

- F0 animals: For 10 weeks before pairing until termination after litters were weaned.
- F1 animals: From weaning for minimum of 10 weeks before pairing and until termination after litters were weaned. Direct treatment of selected F1 offspring commenced at weaning (Day 21 of age). The unselected F1 offspring, and the F2 offspring received no direct treatment but may have been exposed in utero or via the milk.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 Control (corn oil)

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Group 2 (test item)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 3 (test item)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 4 (test item)

No. of animals per sex per dose:

28

Control animals:

yes

Details on study design:

- DOSE SELECTION RATIONALE:
Dose levels of 100, 300 and 1000 mg/kg/day were set following a review of the results of the OECD 421 reproductive screening study conducted with Reaction product of 3,5,5-trimethyl-hexanoic acid and 2-metheylpropanoic acid and pentaerythritol (Huntingdon Life Sciences Study No. OWH0020), and the 13-week toxicity study conducted with Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid (Huntingdon Life Sciences Study No. OWH0019). The highest dose level of 300 mg/kg/day was selected, as a more extended treatment period had the potential to produce greater effects than seen in the OECD 421 reproductive screening study (OWH0020) where the treatment period was only two weeks before pairing. In addition, liver changes which may suggest toxicity were seen on the 13 week toxicity study (OWH0019). Low and intermediate dose levels of 30 and 100 mg/kg/day were selected to give an approximate 3-fold difference between dose levels. 

- RATIONALE FOR ANIMAL ASSIGNMENT (IF NOT RANDOM):
The animals were assigned randomly


- FASTING PERIOD BEFORE BLOOD SAMPLING FOR CLINICAL BIOCHEMISTRY: Yes

- OTHER: N/A

Positive control:

No

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes 

- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes 

- Time schedule: Twice daily. A detailed weekly physical examination was performed once each week for F0 animals and for F1 animals selected for the formal F1 generation to monitor general health

BODY WEIGHT: Yes 

- Time schedule for examinations: F0 males - Day that treatment commenced (Week 0) then each week and at necropsy. F0 females - Day that treatment commenced (Week 0). Each week until mating was detected. Days 0, 7, 14 and 20 after mating. Days 1, 4, 7, 14, 21, 25 and 28 post-partum. F1 animals - At same frequency as F0 animals following selection (nominally 4 weeks of age).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): 

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

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes; F0 males and females: Weekly until paired for mating. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage. F0 females: Days 0-6, 7-13 and 14-19 after mating and Days 1-3, 4-6, 7-13 and 14-20 of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal. F1 animals: Recorded at the same frequency as F0 animals following selection, at approximately 4 weeks of age.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not stated

 - Time schedule for examinations:
 


OTHER:
- Mortality: Near the start and end of each working day
- Parturition observations and gestation length :
Duration of gestation - Time that elapsed between mating and commencement of parturition.
Parturition observations - From Day 20 after mating animals were checked three times daily for evidence of parturition. The progress and completion of parturition was monitored; numbers of live and dead offspring were recorded and any difficulties observed were noted.

Oestrous cyclicity (parental animals):

Dry smears:
Smears were taken daily for 22 days before pairing, using cotton swabs moistened with saline. The smears were subsequently examined to establish the duration and regularity of the oestrous cycle.

Wet smears:
After pairing with the male, daily smearing was continued using pipette lavage, until evidence of mating was observed.
For four days before scheduled termination (nominally Days 25 to 28 post partum) daily vaginal smears were taken and used to determine the stage of the oestrous cycle at termination.
For females whose litters had previously died, smears were taken on a theoretical
Days 25-28. Females that failed to litter or mate were retained and smeared for four days starting on the day on which the first batch of ‘true’ Day 25 females started smearing, then killed with that first batch of females.

Sperm parameters (parental animals):

Parameters examined in [all/F0/F1] male parental generations:
- The left vas deferens, epididymis and testis was removed and the epididymis and testis were weighed
- Sperm motility – all groups: The percentages of motile and progressively motile sperm were reported. The sample for Group 2 male 346 was not analysed due to a system error.
- Sperm count – Groups 1 and 4
- Sperm morphology – Groups 1 and 4: At least 200 sperm were assessed for each male of groups 1 and 4. The percentages of normal sperm and abnormal sperm were reported.
- Homogenisation-resistant spermatids count – Groups 1 and 4

Litter observations:

Clinical signs: All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter.

Offspring identification: On Day 1 of age each offspring was numbered individually within each litter using a toe tattoo.

Litter size : Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age. On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter.

Sex ratio of each litter: Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.

Individual offspring bodyweights: Recorded on Days 1, 4 (before culling), 7, 14, 21 and 25 of age.

Weaning of offspring (selected F1 generation): The dam was removed from the litter cage and selected offspring were weaned on Day 21 of age. Unselected offspring were killed on Day 21 of age.

Selection of offspring (F1 generation): The selection of offspring to form the F1 generation was made on Day 21 of age, and selected F1 animals separated from littermates on Day 21 of age.

Postmortem examinations (parental animals):

F0/F1 generation adults
Scheduled kill - males : When the majority of litters had weaned (after 18 Weeks of treatment for the F0 males or 18 weeks of the F1 generation for the F1 males).
Scheduled kill - females: Day 28 post partum. Females failing to mate or produce viable litter were retained and smeared with first females reaching Day 25 post-partum before scheduled kill with the same first females reaching Day 28 post-partum. Healthy females with total litter loss were retained and killed on Day 28 post-partum (despite the litter loss).
Uterus: Number of implantation sites.

Postmortem examinations (offspring):

F1/F2 generation offspring (unselected)
Culls: On day 4 of age

F1 and F2 offspring were subject to a complete macroscopic examination. Additionally, the following procedures were applicable:
- Premature deaths (before weaning) Missing offspring and any grossly autolysed or grossly cannibalised could not be examined. All other remaining offspring dying before weaning were examined as detailed above; the examination also included an assessment for the presence of milk in the stomach, where this was possible.
- Culled offspring (Day 4): Only offspring that were externally abnormal were examined as detailed above. Offspring considered to be externally normal were discarded without examination.
- Scheduled kill (Day 21of age): One male and one female offspring selected at random from each litter. The bodyweight was recorded and the requisite organs were weighed and fixed. The cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves.

The organs weighed, tissue samples fixed and sections examined microscopically

Statistics:

The following sequence of statistical tests was used for bodyweight, food consumption, litter size and survival indices, sexual maturation and organ weight data: Bartlett's test, Dunnett's test, Shirley's test and Williams’ test
For litter size and survival indices and sexual maturation data: Fisher’s Exact tests
Sex ratio were analysed by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect: Wald chi-square test was used
For F0 oestrous cycles an exact one-tailed (upper-tail) Linear-by-linear test was performed
For F1 gestation length and sperm counts an exact two-tailed Linear-by-linear test

For F1 oestrous cycles an exact one-tailed (upper-tail) Linear-by-linear test
For F1 number mating, number conceiving, number fertile and number live an exact one- tailed (lower-tail) Cochran-Armitage test was applied

Reproductive indices:

Male: Sexual maturation was assessed by daily examination from Day 38 of age until balano-preputial separation occurred. Bodyweight was recorded on the day of completion of separation.
Female: Sexual maturation was assessed by daily examination from Day 28 of age until vaginal opening occurred. Bodyweight was recorded on the day of vaginal opening.

Offspring viability indices:

A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. Other index included, Post implantation survival index, Live birth index, lactaing index

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were five mortalities, none of which were considered to be related to treatment:
One Group 4 male (animal number 104) was found dead on Day 82 of the study, there were no signs or abnormalities noted.
Three Group 3 females (animal numbers 171, 182, 196) and one Group 4 female (animal number 221) were killed for welfare reasons.
- Animal 171 was killed on Day 23 of gestation (around the time of parturition) due to clinical signs of piloerection, dull eyes, abnormal gait, hunched posture and pallor. Macroscopicand microscopic observations included the presence of two late resorptions with placentae in the vagina, masses in the left kidney, and an enlarged spleen. The liver also had increased sinusoidal leukocytes (leukocytosis) and there was marked extramedullary haematopoiesis in the spleen. The findings are consistent with septicemia, probably arising from the endometritis in the uterus. This is considered the main factor contributing to morbidity.
-Animal 182 was killed on Day 23 of gestation shortly after completion of parturition due to clinical signs of piloerection, pallor and dark staining around the vagina. Gross abnormalities included pallor of the adrenals and kidneys, dehydrated, dark contents of the caecum, and red staining of the perigenital fur. Microscopic examination showed moderate hyperplasia of the uterine endometrium with luminal haemorrhage. There was no evidence of a decidual response in the uterus. The main factor contributing to morbidity was uterine haemorrhage.
-Animal 196 was killed on Day 1 of lactation due to clinical signs of deep and slow respiration, piloerection, blood around the vagina, hunched posture, pallor and general poor clinical condition. Macroscopic abnormalities included a thickened uterus, reduced contents of the large intestine, fluid in the abdominal cavity and red staining of the perigenital fur. Microscopic findings included inflammation of the uterine endometrium (endometritis) with luminal haemorrhage. A decidual response with necrosis and inflammation was present in the uterine wall representing resorption sites or post parturition tissue repair. The main factors contributing to morbidity were uterine inflammation and haemorrhage.
-Animal 221 was killed on Day 22 of gestation (around the time of parturition) due to clinical signs of underactivity, reduced body temperature, piloerection, hunched posture and general poor clinical condition. Macroscopic and microscopic findings included the presence of a placenta in the vagina, small spleen and red stained perigenital fur, inflammation of the uterus (endometritis) with luminal haemorrhage. The main factors contributing to morbidity were uterine inflammation and haemorrhage.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no effects of the test item on the reproductive tract tissues of F0 males or females dosed with Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid. Changes related to treatment with the test item were seen in the kidneys of males dosed with 100 and 300 mg/kg/day.
Incidental findings included.

Seminiferous tubules were evaluated with respect to their stage in the Spermatogenic cycle and the integrity of the various cell types present within the different stages.

No cell or stage abnormalities were noted.

The ovary, uterus and vagina were evaluated for morphological synchronization of the stage of the estrous cycle in each tissue. Any asynchrony of cycling or any abnormalities in morphology were recorded.
All other histological changes were considered to be incidental and unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles, pre-coital interval, mating performance and fertility and gestation length and gestation index were considered to be unaffected by treatment with Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

No adverse effects on sperm motility, concentration or morphology were apparent after treatment with Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid at doses up to 300 mg/kg/day.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

The pairings described below were identified as having suspect fertility. Macroscopic abnormalities were recorded for all tissues;
Mating of Female 115 with Male 3 (Group 1): Although there were no macroscopic abnormalities in either animal, sparse corpora lutea were noted microscopically in the ovary of the female. Therefore, suspect fertility was considered female mediated and related to the reduction in corpora lutea.

Mating of Female 121 with Male 9 (Group 1): the male exhibited microscopically small testes and both epididymides which correlated with severe atrophy of the seminiferous tubules in the testis and severe reduction in epididymal sperm. Therefore, the suspect fertility was considered likely to be male mediated and related to the testicular tubular atrophy.

Mating of Female 158 with Male 46 (Group 2): There were no macroscopic or microscopic abnormalities in either animal. The cause of the suspect fertility is undetermined.

Mating of Female 192 with Male 80 (Group 3): The left lobe of the thyroid of the male was macroscopically small but there was no microscopic correlation and there were no other microscopic abnormalities in any of the tissues examined. The cause of the suspect fertility is undetermined.

Mating of Female 204 with Male 92 (Group 4): The male had a few scabs on the skin of the head and the female exhibited a few depressions on the kidney (no microscopic correlate) and the lung had pale areas on the lung which correlated microscopically with minimal aggregates of alveolar macrophages. Consequently the cause of the suspect fertility is undetermined.

For Control animals and animals receiving 30, 100 and 300 mg/kg/day separation from the parent female at weaning was well tolerated and was not associated with any clinical signs or decline in condition.

The number of implantations and litter size and offspring survival through to weaning in treated groups were similar to Control values and were unaffected by treatment. Sex ratios were unaffected by treatment with Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid, and did not indicate a selective effect of sex on survival of the offspring to weaning.

The mean bodyweights of male and female offspring on Day 1 of age in the 300 or 100 mg/kg/day groups were marginally lower than in Controls; differences attained statistical significance for females in both groups and male in the 300 mg/kg/day group only. There was no effect of treatment on the subsequent bodyweight gains of these offspring and bodyweights at weaning on Day 21 of age were similar to Controls.

Intergroup differences in organ weights for male and female offspring in Groups 1, 2, 3 and 4 on Day 21 of age did not indicate any effect of maternal treatment.

Necropsy of offspring dying before weaning showed absence of milk in the stomach as the predominant finding. This is a common observation for offspring dying at such a young age and was not considered to indicate any adverse effect of treatment. There were no treatment related findings among offspring examined at scheduled termination on Day 21 of age.

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs or signs in association with treatment with Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid seen throughout treatment of the F1 parental animals

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

- One Group 3 male (animal number 363) was found dead on Day 52 of the study, there were no clinical signs or microscopic evidence of death. Macroscopically the oesophagus contained food and the lungs were dark.
- Animal number 441 was killed on Day 17 of gestation due to clinical signs of piloerection, dull eyes, hunched posture, pallor and bloody discharge from the vagina. This animal was found to be pregnant at macroscopic examination with eight early resorptions in the left uterine horn and seven early resorptions in the left uterine horn. Macroscopic findings included dark fluid in the uterus and vagina and pallor of various tissues. Microscopically, there was luminal haemorrhage in the uterus and vagina. A decidual response was present in the wall of the uterus. The main factor contributing to morbidity was uterine haemorrhage.
- Animal number 460 was killed on Day 7 of lactation due to general poor clinical condition such as reduced body temperature, thin build and hunched posture. Macroscopic findings included dark contents in the intestines, and a small thymus. There were no microscopic findings that were considered contributory to death.
- Animal number 468 was killed on Day 14 of gestation after the rupturing of a firm, moveable, subcutaneous swollen area on the upper ventral thorax. This animal was found to be pregnant at macroscopic examination with nine early resorptions in each uterine horn. Gross findings included a mass in the thoracic mammary gland associated with scabbing of the overlying skin. Microscopically the mass correlated with a mammary adenocarcinoma. A decidual response was present in the uterus. The mammary adenocarcinoma was the main factor contributing to morbidity.
- Animal number 486 was killed on Day 2 of lactation. There were no significant macroscopic observations. Microscopic findings included inflammation of the uterus (endometritis). This was the main factor contributing to morbidity.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no effect of treatment with Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid on the bodyweight or bodyweight gain of F1 males throughout the treatment period

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment with Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid on the food consumption of F1 females throughout gestation and lactation.

Food efficiency:

no effects observed

Description (incidence and severity):

There was no effect of treatment with Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid on the food conversion efficiency.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The following effects on organ weights were apparent:
At termination, the group mean absolute and bodyweight relative kidney weights were statistically significantly high (p≤0.01) for males treated with Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid at 100 and 300 mg/kg/day and the group mean absolute and bodyweight relative liver weights were statistically significantly high (p≤0.01) for males which received 300 mg/kg/day when compared with the controls. In addition, the group mean absolute adrenal weights were statistically significantly high for males treated with tets item at 300 mg/kg/day, and group mean absolute and bodyweight relative spleen weights were statistically significantly high for males treated with test item at 300 mg/kg/day.

In females there was a statistically significant increase (p≤0.01) in absolute and bodyweight relative liver weights in females which received Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid at 300 mg/kg/day, and a statistically significant increase in absolute (p≤0.01) and bodyweight relative (p≤0.05) ovary weights in females which received test item at 300 mg/kg/day. The group mean liver weight relative to bodyweight for females treated at 100 mg/kg/day was statistically significantly higher (p≤0.05) than that of the control goup.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination revealed no test substance related lesions.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no effects of test item on the reproductive tract tissues of F1 males or females dosed with test item. Changes related to treatment with test item were seen in the kidneys of males dosed with 100 and 300 mg/kg/day that were examined due to macroscopic abnormalities. The changes associated with the hyaline droplet nephropathy were similar in nature and severity to those seen in the F0 generation. The kidneys of ten males dosed with 300 and three males dosed with 100 mg/kg/day were examined and showed changes. Hyaline droplets were not present in the kidneys of the three controls and the six Group 2 males examined.

Other effects:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment at 30, 100 or 300 mg/kg/day on the time of completion of balano preputial separation for F1 males or on the time of completion of vaginal opening for females when compared with the time of completion for the control group.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

There was no effect of treatment on the numbers of primordial ovarian follicles counted for the F1 adults in the 300 mg/kg/day group when compare with the controls.

The pairings described below were identified as having suspect fertility. Macroscopic abnormalities were recorded for all tissues. The reproductive tissues were examined microscopically.

Mating of Female 450 with Male 358 (Group 3): Macroscopically, there were no signs of implantation in the uterus. This was confirmed by microscopic examination. In the female, pelvic dilatation was noted macroscopically and microscopically and minimal chronic progressive nephropathy was also present in the kidney. Occasional atrophic tubules were present in the right testis of the male. This is a common background finding in rats and would not be responsible for decreased fertility. There were no other macroscopic or microscopic abnormalities in either animal. The cause of the suspect fertility is undetermined.

Mating of Female 451 with Male 359 (Group 3): In the female, the ovaries were macroscopically small and pale. Microscopically, the ovaries contained follicular cysts and no corpora lutea. The uterus had no signs of implantation by macroscopic and microscopic examination. There were no other microscopic abnormalities in any of the tissues examined. The suspect fertility is likely female mediated and related to the presence of follicular cysts and absence of corpora lutea.

Mating of Female 445 with Male 353 (Group 3): There were no macroscopic or microscopic signs of implantation in the uterus. There were no other macroscopic or microscopic abnormalities in either animal. The cause of the suspect fertility is undetermined.

Mating of Female 463 with Male 371 (Group 3): There were no macroscopic or microscopic signs of pregnancy in the uterus. In the male minimal lymphoid aggregates were present microscopically in the prostate. There were no other macroscopic or microscopic abnormalities in either animal. The cause of the suspect fertility is undetermined.

Mating of Female 485 with Male 393 (Group 4): There were no macroscopic or microscopic signs of implantation in the uterus. Both testes were macroscopically small and flaccid and the left epididymis was macroscopically small. The findings correlated microscopically with severe atrophy of the seminiferous tubules in the testis and severe reduction in epididymal sperm. Macroscopic observations of multiple depressions in the kidneys of the male correlated microscopically with slight chronic progressive nephropathy and hyaline droplets in the cortical tubules. There were no other macroscopic or microscopic abnormalities in any of the tissues examined. The suspect fertility is likely male mediated and related to the testicular tubular atrophy.

Mating of Female 491 with Male 375 (Group 4): There were no macroscopic or microscopic signs of pregnancy in the uterus. In the female, macroscopic observations of pale areas on the adrenals and depressions on the kidney had no microscopic correlates. In the male, there was macroscopic hair loss on the side of the face (not examined microscopically) and minimal lymphoid aggregates and focal neutrophilic infiltrate present microscopically in the prostate. There were no other macroscopic or microscopic abnormalities in either animal. The cause of the suspect fertility is undetermined.

For Control animals and animals receiving 30, 100 and 300 mg/kg/day separation from the parent female at weaning was well tolerated and was not associated with any clinical signs or decline in condition. The number of implantations and litter size in Groups 1, 2, 3 and 4 were similar to Control values and were unaffected by treatment and the extent of offspring survival through to weaning in Groups 1, 2 and 3 were similar to Control values and were unaffected by treatment.

The absolute brain weight of male offspring in Group 4 (parental treatment at 300 mg/kg/day) was statistically significantly lower than that of the Control (p≤0.05), however, in the absence of an effect on the brain weight relative to bodyweight, and in the absence of a similar effect on female offspring, this difference is considered not related to parental treatment with the test item at 300 mg/kg/day. There were no treatment related findings among offspring examined at scheduled termination on Day 21 of age.

Bodyweight on Day 1 of age and subsequent bodyweight gains to weaning on Day 21 of age were unaffected by treatment with the test item at 30 and 100 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The extent of offspring survival between Day 1 and Day 4 of age in the 300 mg/kg/day group as indicated by the viability index (%) was statistically significantly lower than that of the the Control. This was due to a viability index of 95% or less in 10/22 litters in Group 4, compared with 3/24 litters in the Control group, 0/23 litters in Group 2 and 2/19 litters in Group 3.The extent of offspring survival from Day 4 of age through to weaning (the lactation index) in Group 4 was similar to Control values and considered unaffected by treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweight of both male and female offspring on Day 1 of age was statistically significantly lower than that of the Control (p≤0.05) following maternal treatment with the test item at 300 mg/kg/day. Subsequent bodyweight gains to weaning on Day 21 of age were unaffected by treatment with Tetraesters of pentaerythritol with 2- methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid at 300 mg/kg/day. Bodyweight on Day 1 of age and subsequent bodyweight gains to weaning on Day 21 of age were unaffected by treatment with the test item at 30 and 100 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute brain weight of male offspring in Group 4 (parental treatment at 300 mg/kg/day) was statistically significantly lower than that of the Control (p≤0.05), however, in the absence of an effect on the brain weight relative to bodyweight, and in the absence of a similar effect on female offspring, this difference is considered not related to parental treatment with the test item at 300 mg/kg/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Necropsy of offspring dying before weaning showed absence of milk in the stomach as the predominant finding. This is a common observation for offspring dying at such a young age and was not considered to indicate any adverse affect of dietary exposure. There were no treatment related findings among offspring examined at scheduled termination on Day 21 of age.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

Treatment related:

no

Conclusions:

Based on the results of this study it was concluded that the no-observed-adverse-effect-level (NOAEL) for general toxicity and reproductive performance of the F0 and F1 adult animals is 300 mg/kg/day. The NOAEL for offspring survival and growth is 100 mg/kg/day, based on reduced survival of the F2 offspring between Day 1 and 4 of age, and the low Day 1 bodyweights of the offspring on both generations at 300 mg/kg/day.

Executive summary:

OECD 416 study (2013) - The influence of Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid on the integrity and performance of the male and female reproductive systems, and the growth and development of the offspring, was assessed, when administered continuously by oral gavage through two successive generations of Crl:CD (SD) rats. For the F0 generation, three groups of 28 male and 28 female rats received oral garage dose of 30, 100 or 300 mg/kg/day for ten weeks before pairing, throughout pairing, gestation, lactation and until termination. Similarly constituted Control group received the vehicle (corn oil) by oral gavage for the same duration, while F1 generation comprised of 24 male and 24 female progeny from each group, and they continued to receive the the test item by oral gavage, as per the F0 generation, throughout the same period as for the F0 generation until termination after the litters were weaned. The unselected F1 offspring and the F2 offspring were not treated directly and were terminated at the point of weaning (Day 21 of age).

Clinical condition, bodyweight, food consumption, oestrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance were observed during the course of the study. Seminology for the males, organ weight, macroscopic and microscopic pathology investigations were undertaken on each adult generation. The clinical condition of offspring, litter size and survival, sex ratio, sexual maturation (selected F1 generation only) and bodyweight gain was assessed and organ weight and macroscopic pathology investigations were undertaken on each generation. Sperm parameters for F0 and F1 males were unaffected and the ovarian primordial follicle counts in the F1 females receiving 300 mg/kg/day were unaffected by treatment. Furthermore, the mean age at vaginal opening for F1 females and the mean age at balano-preputial separation of F1 males were also unaffected.

There was no evidence of any adverse effect on clinical condition, bodyweight, bodyweight change, food consumption or food conversion efficiency in the F0 or F1 generations at all dose groups. Reproductive performance of F0 and F1 adults  such as oestrous cycles, mating performance, pre-coital interval, gestation length, litter size and subsequent offspring clinical condition and sex ratio were unaffected. The survival of of F1 offspring was unaffected but the the viability index (%) of F2 was statistically significantly lower  in the high dose groups and a relationship to parental treatment with the test item could not be ruled out. The bodyweight of the F1 and F2 offspring on Day 1 of age was low in the 300 mg/kg/day group, but subsequent weight gain was unaffected by treatment.

There were no necropsy findings among F0 and F1 adults which were considered to be related to treatment. However, macroscopic abnormalities in the form of hyaline droplet nephropathy were observed in the kidneys of F0 or F1 males in the 100 or 300 mg/kg/day groups.

Based on the results of this study it was concluded that the no-observed-adverse-effect-level (NOAEL) for general toxicity and reproductive performance of the F0 and F1 adult animals is 300 mg/kg/day. The NOAEL for offspring survival and growth is 100 mg/kg/day, based on reduced survival of the F2 offspring between Day 1 and 4 of age, and the low Day 1 bodyweights of the offspring on both generations at 300 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The endpoint is concluded based on a single key study with a Klimish rating of 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 421 (2012) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 421), Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid was administered to 10 male and 10 female Crl:CD(SD) strain rats in corn oil via oral gavage at dose of 100, 300 and 1000 mg/kg/day for up to 5 weeks. In addition, 10 male and 10 female control rats were administered corn oil only. 

Systemic toxicity (F0): There were three deaths all in females at the end of gestation/early lactation observed at 300 1000 mg/kg/day but necropsy did not reveal test item related effects. Underactivity, irregular breathing, dark discharge from the vagina, partially closed eyelids and skin pallor observed in three female at 300 and 1000 mg/kg/day. 

Bodyweight- There were no clear adverse effects of treatment of upon bodyweights of males or of females before pairing, with treated animals gaining similar amounts of or slightly more weight than controls. There were no clear effects on food consumption in the weeks before pairing or, for females, during gestation and lactation at doses up to 1000 mg/kg/day. 

No toxicologically significant effects were detected in terminal kill animals of either sex treated with the test item.  However, Changes of an uncertain relationship to treatment were seen in the ovary of females given the test item at 100, 300 and 1000 mg/kg/day. Prominent corpora lutea of pregnancy in the ovaries were more frequently seen in all treated groups than in controls, particularly in females dosed at 1000 mg/kg/day. 

There were no effects on reproductive performance, mating, estrous cycle, fertility with all gestation lengths within the expected range of 22 to 23 days. The offspring survival up to Day 7 of age was slightly low for some litters in the treated groups but it was considered that there was no dosage related effect. Offspring bodyweight on Day 1 of age was slightly low (82 % of control) for litters in the highest dose group (1000 mg/kg/day), but by Day 7 of age there was an improvement in relative offspring weight (88/90 % of male/female weight. Birth weight at 300 mg/kg/day was also slightly low (86-88 % of control). 

Therefore, it was considered that treatment at 300 and 1000 mg/kg/day had biologically significant effects upon the in utero growth of the offspring, but there was no evidence of developmental abnormality. There was no evidence of an effect of treatment upon reproductive performance of the parental generation or that the in utero effect would lead to an effect upon reproductive performance of the F1 generation.  It is concluded that that the No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/day for reproductive performance and 100 mg/kg/day was considered to be the overall NOAEL because of the effects seen on birth weight at 300 and 1000 mg/kg/day.  

OECD 416 (2013) - The influence of the test item on the integrity and performance of the male and female reproductive systems, and the growth and development of the offspring, was assessed, when administered continuously by oral gavage through two successive generations of Crl:CD (SD) rats. For the F0 generation, three groups of 28 male and 28 female rats received oral garage dose of 30, 100 or 300 mg/kg/day for ten weeks before pairing, throughout pairing, gestation, lactation and until termination. Similarly constituted Control group received the vehicle (corn oil) by oral gavage for the same duration, while F1 generation comprised of 24 male and 24 female progeny from each group, and they continued to receive the test item by oral gavage, as per the F0 generation, throughout the same period as for the F0 generation until termination after the litters were weaned. The unselected F1 offspring and the F2 offspring were not treated directly and were terminated at the point of weaning (Day 21 of age).

There was no evidence of any adverse effect on clinical condition, bodyweight, bodyweight change, food consumption or food conversion efficiency in the F0 or F1 generations at all dose groups. Reproductive performance of F0 and F1 adults such as oestrous cycles, mating performance, pre-coital interval, gestation length, litter size and subsequent offspring clinical condition and sex ratio were unaffected. The survival of F1 offspring was unaffected but the viability index (%) of F2 was statistically significantly lower in the high dose groups and a relationship to parental treatment with the test item could not be ruled out. The bodyweight of the F1 and F2 offspring on Day 1 of age was low in the 300 mg/kg/day group, but subsequent weight gain was unaffected by treatment.

Sperm parameters for F0 and F1 males were unaffected and the ovarian primordial follicle counts in the F1 females receiving 300 mg/kg/day were unaffected by treatment. Furthermore, the mean age at vaginal opening for F1 females and the mean age at balano-preputial separation of F1 males were also unaffected.

Abnormalities in the form of hyaline droplet nephropathy were observed in the kidneys of F0 or F1 males in the 100 or 300 mg/kg/day groups.

Based on the results of this study it was concluded that the no-observed-adverse-effect-level (NOAEL) for general toxicity and reproductive performance of the F0 and F1 adult animals is 300 mg/kg/day. The NOAEL for offspring survival and growth is 100 mg/kg/day, based on reduced survival of the F2 offspring between Day 1 and 4 of age, and the low Day 1 body weights of the offspring on both generations at 300 mg/kg/day.

Effects on developmental toxicity

Description of key information

NOAEL (Developmental toxicity) =  1000 mg/kg bw/day; OECD 414; Armour G. (2012)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 May 2012 - 4 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL

- Source and lot/batch No.of test material: JA01YX10
- Expiration date of the lot/batch: Jan 2013
- Purity test date: 95.8 %



RADIOLABELLING INFORMATION (if applicable): N/A



STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL

- Storage condition of test material:bRoom temperature (ca 20 °C), in the dark under nitrogen
- Stability under test conditions: Stable

- Solubility and stability of the test substance in the solvent/vehicle: stable in the liquid matrix at concentrations of 2 and 200 mg/mL for 24 hours at ambient temperature and for up to 15 days when refrigerated (nominally 2 to 8C). 

- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A



TREATMENT OF TEST MATERIAL PRIOR TO TESTING

- Treatment of test material prior to testing: The test material was used as supplied. The test item was prepared for administration as a series of graded concentrations in the vehicle - corn oil.
- Preliminary purification step (if any): Dose range finder was conducted prior to the main experiment ignorer to determine suitable dose levels.

- Final dilution of a dissolved solid, stock liquid or gel: 5 mL/kg
- Final preparation of a solid: N/A



FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A

Species:

rat

Strain:

Crj: CD(SD)

Remarks:

Crl:CD(SD) rats

Details on test animals or test system and environmental conditions:

TEST ANIMALS

- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: [yes/no]: Yes
- Age at study initiation: 10 weeks
- Weight at study initiation: Weight range 228 to 283 g.
- Fasting period before study: Not Stated
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.



DETAILS OF FOOD AND WATER QUALITY:

- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet.
- Water (e.g. ad libitum): Polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 40 to 70 %
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.

- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

IN-LIFE DATES: From: To: 20 June 2012 - 19 July 2012

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Females were treated from Day 6 to Day 19 (inclusive) after mating.
Animals received the test material or vehicle control formulations orally at a volume-dose of 5 mL/kg bodyweight, using a suitably graduated syringe and a rubber catheter inserted via the mouth into the stomach.
All animals were dosed in sequence of cage-number within each group, once each day at approximately the same time each day, seven days per week. The volume administered to each animal was calculated from the most recently recorded scheduled bodyweight.
A daily record of the weight of each formulation dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Approximately 50 % of the final volume of vehicle was added to the test item and magnetically stirred until the test material had dispersed and homogenous. Specimen formulations were prepared at concentrations of 2 mg/mL and 200 mg/mL .

The stability was assessed following storage at ambient temperature (nominally 21 °C) for 0, 2 and 4 hours and 1 day, and refrigeration (nominally 2 to 8 ºC) for 1 day, 8 days and 15 days.

Single samples were taken for assay from the top, middle and bottom of the magnetically stirred formulation and homogeneity was determined by analysis of these samples. Stability was determined from the mean concentration of the test item in the vehicle at each sampling point.

The results of the analysis confirmed that the test item produced an homogenous suspension and was stable in the liquid matrix at concentrations of 2 and 200 mg/mL for 24 hours at ambient temperature and for up to 15 days when refrigerated (nominally 2 to 8 ºC).

Details on mating procedure:

The ainimals were paired on a 1:1 basis with stock males from the same source. Daily checks were made after pairing for evidence of mating, including ejected copulation plugs in cage trays and the presence of sperm in a vaginal smear.
Animals were allocated to study on Day 0 of gestation, when positive evidence of mating was detected. Only females with a sperm positive vaginal smear or at least two copulation plugs were selected. Females were allocated to group and cage position in sequence of mating, thus ensuring that animals mated on any one day were evenly distributed amongst the groups. The sequence of allocation was controlled to prevent stock males from providing more than one mated female in each treatment group.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Once daily

Duration of test:

Day 6 to Day 19 after mating

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 Control (Corn oil)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 (test item)

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3 (test item)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4 (test item)

No. of animals per sex per dose:

20

Control animals:

yes

Details on study design:

The oral gavage route of administration was chosen to simulate a condition of potential human exposure. The doses used (0, 100, 300 and 1000 mg/kg/day) were selected in conjunction with the Sponsor following a review of the results of a OECD 421 reproductive screening study using this compound performed in these laboratories (Huntingdon Life Sciences Report Number: OWH0020). It was expected that 100 mg/kg/day is the NOEL (no-observed-effect-level), and that there was the possibility of effects upon fetal weight at 300 mg/kg/day, and reduced fetal weight at 1000 mg/kg/day. The test substance was administered randomly assinged animals from Day 6 to 19 after mating.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: Yes 

- Time schedule: Twice daily


BODY WEIGHT: Yes 

- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.



FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes 

- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive.

 

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

 


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20

- Organs examined: yes
 


OTHER:
In addition, a more detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

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
The gravid uterus was weighed; this weight included the weight of the cervix and ovaries.

Fetal examinations:

All fetuses and placentae were dissected from the uterus and weighed individually. Fetuses were individually identified within the litter, using a coding system based on their position in the uterus. Each fetus and placenta was externally examined and any abnormalities were recorded. The sex of each fetus was recorded.
Approximately half of the fetuses in each litter were eviscerated and their skeletons were fixed in Industrial Methylated Spirit prior to processing and staining with Alizarin Red. The remaining fetuses were fixed whole in Bouin’s fluid.

Statistics:

The following sequence of statistical tests was used for bodyweight, food consumption, organ weights and litter data: Bartlett's test, Dunnett's test, Shirley's test and Williams’ test
For gravid uterine weight, litter size and survival indices and fetal, placental and litter weight data, if 75 % of the data (across all groups) were the same value, for example c, Fisher’s Exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

Pre/post implantation loss and sex ratio were analysed by generalised mixed linear model with binomial errors, a logit link function and litter as a random effect (Lipsitz et al 1991). Each treated group was compared to control using a Wald chi-square test. For pre-implantation loss, the numerator was Number of corpora lutea - Number of implantations, the denominator was Number of corpora lutea. For post-implantation loss, the numerator was Number of implantations - Number of live fetuses, the denominator was Number of implantations. For sex ratio, the numerator was Number of males, the denominator was Number of live fetuses.
For resorptions, each treated group was compared to control by exact Wilcoxon rank sum test (Wilcoxon 1945).

Significant differences between Control and treated groups were expressed at the
5% (p<0.05) or 1% (p<0.01) level. The key to the annotation used on the tables that contain statistical results is given below.
Av Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.
Sh Treated groups compared to Control using Shirley’s test.
Wa Treated groups compared to Control using Wald’s test
Wc Treated groups compared to Control using Wilcoxon rank sum test Wi Treated groups compared to Control using Williams’ test.
* p<0.05 ** p<0.01

Indices:

Findings observed were classified, according to severity and incidence, as:
Major abnormalities: normally rare, definitely detrimental to normal subsequent development, possibly lethal, e.g. ventricular septal defect
Minor abnormalities: minor differences from normal that are detected relatively frequently considered to have little detrimental effect and may be a transient stage in development e.g. bipartite centrum, dilated ureter.
Variants: alternative structures or stages of development occuring regularly in the control population, e.g. number of ribs, incomplete ossification of 5th and 6th sternebrae.

Historical control data:

Included in the report

Clinical signs:

no effects observed

Description (incidence and severity):

Signs seen in association with dosing included occasional chin rubbing for some females in all groups throughout gestation and for most females receiving the test item at 1000 mg/kg/day towards the end of gestation. No other signs in association with dosing were recorded.

Dermal irritation (if dermal study):

not examined

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

clinical signs

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day there was a slightly higher incidence of incompletely ossified/ unossified cranial centres, hyoid, and pelvic bones compared with concurrent controls. These are all within historical control data levels.

There was also a low incidence of folded retinas. In the absence of any related findings, this is also considered not to be treatment related.

At 300 mg/kg/day there was a slightly higher incidence of incompletely ossified/ unossified cranial centres compared with concurrent controls. This is within historical control data levels.

Visceral malformations:

no effects observed

Other effects:

not specified

Key result

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 effect observed

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Tables containing raw data are attached in full study report.

Conclusions:

Oral administration of the test item to pregnant CD rats during the organogenesis and fetal growth phase of gestation resulted in slight maternal toxicity at 1000 mg/kg/day while embyro-fetal survival, growth and development were considered not affected. Fetal and litter weight of females which had received the test item at 1000 mg/kg/day were slightly, but statistically significantly lower than the weight of the concurrent control (approximately 95 % of control values). Offspring birth weight in females treated throughout pregnancy (HLS study OWH0020) were much more severely affected and were nearly 20 % below control values, suggesting that continued treatment at this level could be damaging to late fetal growth.

Based on the condition of the study, it was concluded that the no-observed-adverse-effect-level (NOAEL) for both maternal toxicity and embryo-fetal development was 1000 mg/kg/day.

Executive summary:

OECD 414 (2012) - In Embryo-Fetal Development al toxicity study (OECD 414), Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid was administered to  20 female Crl:CD(SD) strain rats  during the organogenesis and fetal growth phases of pregnancy via oral gavage at dose of 100, 300 and 1000 mg/kg/dayfrom Day 6 to Day 19 after mating. In addition,  a group of 20 female control rats were administered corn oil only at the same dose volume throughout the same period. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination. A summary of adult responses to the test item are described below; Systemic toxicity (maternal)

 

Mortality- There were no deaths and no clinical signs seen considered related to treatment with the test item.

Clinical signs- occasional chin rubbing for some females in all groups throughout gestation and for most females receiving the test item at 1000 mg/kg/day towards the end of gestation. No other signs in association with dosing were recorded.  

Bodyweight - There were no maternal bodyweight or bodyweight gain.

Food consumption and efficiency - Food consumption for females receiving the test item at 100, 300 or 1000 mg/kg/day was generally similar to that of the Control throughout gestation but the food consumption of females receiving the test item at 1000 mg/kg/day was slightly lower than that of the Control between Days 18-19 of gestation.

  

Histopathological changes and Necropsy - There were no macroscopic findings at necropsy which were considered related to treatment with the test item from Day 6 to Day 19 after mating.

  

Organ weights - Values for the weight of the gravid uterus, adjusted bodyweight and adjusted bodyweight change at dose levels up to 1000 mg/kg/day were similar to control values and considered unaffected by treatment with the test item.

All females were found to be pregnant at necropsy. Litter data as assessed by the mean numbers of corpora lutea, implantations, embryo-fetal resorptions, live young, sex ratio and the extent of pre- and post-implantation loss was similar across all groups.

Developmental toxicity (Fetal)

Embyro-fetal survival, growth and development were considered not affected by treatment at dose levels up to 1000 mg/kg/day

Fetal and litter weight of females which had received the test item at 1000 mg/kg/day were marginally lower than the weight of the concurrent control (approximately 95 % of control values, statistically significant for overall fetal weight and male fetuses). Offspring birth weight in females treated throughout pregnancy (HLS study OWH0020) were much more severely affected and were nearly 20 % below control values, suggesting that continued treatment at this level could be damaging to late fetal growth.

There were incidence of major and minor abnormalities and skeletal variants without any relationship to treatment. This included, a slightly higher incidence of incompletely ossified/ unossified cranial centres, hyoid, and pelvic bones compared with concurrent controls at 1000 mg/kg/day. At this dose, there was also a low incidence of folded retinas, however, in the absence of any related findings, it was considered not to be treatment related.  At 300 mg/kg/day there was a slightly higher incidence of incompletely ossified/ unossified cranial centres compared with concurrent controls. This is within historical control data levels.

Based on the results of this study, it is concluded that the no-observed-adverse-effect-level (NOAEL) for both maternal toxicity and embryo-fetal development was 1000 mg/kg/day.

The study was acceptable and satisfies the guideline requirements for an OECD 414 in the rat.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The endpoint is concluded based on a single key study with a Klimish rating of 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

OECD 414 (2012) - In Embryo-Fetal Development al toxicity study (OECD 414), the test item was administered to 20 female Crl:CD(SD) strain rats during the organogenesis and fetal growth phases of pregnancy via oral gavage at dose of 100, 300 and 1000 mg/kg/dayfrom Day 6 to Day 19 after mating. In addition, a group of 20 female control rats were administered corn oil only at the same dose volume throughout the same period. A summary of adult responses to the test item are described below; Systemic toxicity (maternal). There were no deaths and no clinical signs seen considered related to treatment with Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid, There were no effects on maternal bodyweight or bodyweight gain and food consumption was not affected.

There were no effects on organ weight or any macroscopic findings at necropsy which were considered related to treatment with the test item from Day 6 to Day 19 after mating.  All females were found to be pregnant at necropsy. Litter data as assessed by the mean numbers of corpora lutea, implantations, embryo-fetal resorptions, live young, sex ratio and the extent of pre- and post-implantation loss was similar across all groups.

Developmental toxicity (Fetal): Embyro-fetal survival, growth and development were considered not affected by treatment at dose levels up to 1000 mg/kg/day There were incidence of major and minor abnormalities and skeletal variants without any relationship to treatment. This included, a slightly higher incidence of incompletely ossified/ unossified cranial centres, hyoid, and pelvic bones compared with concurrent controls at 1000 mg/kg/day. At this dose, there was also a low incidence of folded retinas, however, in the absence of any related findings, it was considered not to be treatment related.  At 300 mg/kg/day there was a slightly higher incidence of incompletely ossified/ unossified cranial centres compared with concurrent controls. This is within historical control data levels.

Based on the results of this study, it is concluded that the no-observed-adverse-effect-level (NOAEL) for both maternal toxicity and embryo-fetal development was 1000 mg/kg/day.

Toxicity to reproduction: other studies

Description of key information

NOAEL (systemic toxicity) =  100 mg/kg bw/day; OECD 421; Armour G. (2012)

NOAEL (Reproduction) =  1000 mg/kg bw/day; OECD 421; Armour G. (2012)

NOAEL (F0 and F1) =  300 mg/kg bw/day; OECD 416; Armour G. (2013)

NOAEL (F2) =  100 mg/kg bw/day; OECD 416; Armour G. (2013)

Additional information

OECD 421 (2012) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 421), Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid was administered to 10 male and 10 female Crl:CD(SD) strain rats in corn oil via oral gavage at dose of 100, 300 and 1000 mg/kg/day for up to 5 weeks. In addition, 10 male and 10 female control rats were administered corn oil only. 

Systemic toxicity (F0): There were three deaths all in females at the end of gestation/early lactation observed at 300 1000 mg/kg/day but necropsy did not reveal test item related effects. Underactivity, irregular breathing, dark discharge from the vagina, partially closed eyelids and skin pallor observed in three female at 300 and 1000 mg/kg/day. 

Bodyweight- There were no clear adverse effects of treatment of upon bodyweights of males or of females before pairing, with treated animals gaining similar amounts of or slightly more weight than controls. There were no clear effects on food consumption in the weeks before pairing or, for females, during gestation and lactation at doses up to 1000 mg/kg/day. 

No toxicologically significant effects were detected in terminal kill animals of either sex treated with the test item.  However, Changes of an uncertain relationship to treatment were seen in the ovary of females given the test item at 100, 300 and 1000 mg/kg/day. Prominent corpora lutea of pregnancy in the ovaries were more frequently seen in all treated groups than in controls, particularly in females dosed at 1000 mg/kg/day. 

There were no effects on reproductive performance, mating, estrous cycle, fertility with all gestation lengths within the expected range of 22 to 23 days. The offspring survival up to Day 7 of age was slightly low for some litters in the treated groups but it was considered that there was no dosage related effect. Offspring bodyweight on Day 1 of age was slightly low (82 % of control) for litters in the highest dose group (1000 mg/kg/day), but by Day 7 of age there was an improvement in relative offspring weight (88/90 % of male/female weight. Birth weight at 300 mg/kg/day was also slightly low (86-88 % of control). 

Therefore, it was considered that treatment at 300 and 1000 mg/kg/day had biologically significant effects upon the in utero growth of the offspring, but there was no evidence of developmental abnormality. There was no evidence of an effect of treatment upon reproductive performance of the parental generation or that the in utero effect would lead to an effect upon reproductive performance of the F1 generation.  It is concluded that that the No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/day for reproductive performance and 100 mg/kg/day was considered to be the overall NOAEL because of the effects seen on birth weight at 300 and 1000 mg/kg/day.  

In an OECD 416 study - 2013, the influence of Tetraesters of pentaerythritol with 2-methylpropanoic acid and 3,5,5-trimethyl-hexanoic acid on the integrity and performance of the male and female reproductive systems, and the growth and development of the offspring, was assessed, when administered continuously by oral gavage through two successive generations of Crl:CD (SD) rats. For the F0 generation, three groups of 28 male and 28 female rats received oral garage dose of 30, 100 or 300 mg/kg/day for ten weeks before pairing, throughout pairing, gestation, lactation and until termination. Similarly constituted Control group received the vehicle (corn oil) by oral gavage for the same duration, while F1 generation comprised of 24 male and 24 female progeny from each group, and they continued to receive the the test item by oral gavage, as per the F0 generation, throughout the same period as for the F0 generation until termination after the litters were weaned. The unselected F1 offspring and the F2 offspring were not treated directly and were terminated at the point of weaning (Day 21 of age).

There was no evidence of any adverse effect on clinical condition, bodyweight, bodyweight change, food consumption or food conversion efficiency in the F0 or F1 generations at all dose groups. Reproductive performance of F0 and F1 adults such as oestrous cycles, mating performance, pre-coital interval, gestation length, litter size and subsequent offspring clinical condition and sex ratio were unaffected. The survival of F1 offspring was unaffected but the viability index (%) of F2 was statistically significantly lower in the high dose groups and a relationship to parental treatment with the test item could not be ruled out. The bodyweight of the F1 and F2 offspring on Day 1 of age was low in the 300 mg/kg/day group, but subsequent weight gain was unaffected by treatment.

Sperm parameters for F0 and F1 males were unaffected and the ovarian primordial follicle counts in the F1 females receiving 300 mg/kg/day were unaffected by treatment. Furthermore, the mean age at vaginal opening for F1 females and the mean age at balano-preputial separation of F1 males were also unaffected.

Abnormalities in the form of hyaline droplet nephropathy were observed in the kidneys of F0 or F1 males in the 100 or 300 mg/kg/day groups.

Based on the results of this study it was concluded that the no-observed-adverse-effect-level (NOAEL) for general toxicity and reproductive performance of the F0 and F1 adult animals is 300 mg/kg/day. The NOAEL for offspring survival and growth is 100 mg/kg/day, based on reduced survival of the F2 offspring between Day 1 and 4 of age, and the low Day 1 body weights of the offspring on both generations at 300 mg/kg/day.

Mode of Action Analysis / Human Relevance Framework

Nephrotoxicity in the form of hyaline droplet nephropathy were observed in the kidneys of F0 or F1 males is the main method of toxicity for this substance. However, this is common in rats and not relevant to human.

Justification for classification or non-classification

The substance does not meet the criteria for classification under reproductive toxicity in accordance with GHS and Regulation (EC) No 1272/2008 (CLP).