Fatty acids, C6-12, mixed tetraesters with heptanoic acid, pentaerythritol, 3,5,5-trimethylhexanoic acid and valeric acid

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

451-190-0

EC Name:

-

Cas Number:

156558-98-4

Molecular formula:

C25H44O8 to C43H80O8

IUPAC Name:

3-(heptanoyloxy)-2-[(heptanoyloxy)methyl]-2-[(pentanoyloxy)methyl]propyl octanoate

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

Rationale: This species and strain of rat has been recognized as appropriate for reproduction toxicity studies. WIL Research Europe B.V. has reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Source: F0 Charles River Deutschland, Sulzfeld, Germany.
Age at start: F0-treatment Approximately 11-12 weeks.
Number of F0-animals: 40 females and 40 males.
Acclimatization: F0 At least 5 days prior to start of treatment.
Health inspection: F0 Upon receipt of the animals.
Randomization: F0 Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification: F0 Earmark and tattoo.

Details on mating procedure:

Mating procedures: Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated.
Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes
and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Number of pups: 388 pups.
Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical conditions:
Instrument Alliance Separation Module 2695 (Waters, Milford, MA, USA)
Detector Dual λ Absorbance Detector 2487 (Waters)
Column Symmetry C8, 75 mm × 4.6 mm i.d., dp = 3.5 μm (Waters)
Column temperature 40°C ± 1°C
Injection volume 10 μl
Mobile phase A - acetonitrile
B – water
Gradient
Time [minutes] %A %B
0 70 30
5 100 0
12 100 0
12.1 70 30
15 70 30

Flow 1.0 ml/min
UV detection 210 nm

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
100
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
300
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
1000
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

Mortality / Viability: At least twice daily.
Clinical signs: Daily from treatment onwards up to the day prior to necropsy
Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Oestrous cyclicity (parental animals):

General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Litter observations:

Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe,
were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).
Clinical signs: At least once daily, detailed clinical observations were made for all
animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

The animals were not deprived of food overnight.
All animals surviving to the end of the observation period animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to an external, thoracic and abdominal examination with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

Necropsy was conducted on the following days:
Females which delivered: Lactation Days 5 or 7
Females which failed to deliver1 (nos. 65, 70 and 79): Post-coitum Days 26-27 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).
Females with total litter loss (no. 80): Within 24 hours of litter loss
Males: Following completion of the mating period (a minimum of 28 days of dose administration)

The numbers of former implantation sites and corpora lutea were recorded for all paired females

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation on Days 5 or 7 of lactation.

All pups were sexed and descriptions of all external abnormalities were recorded. At discretion of the Study Director, relevant abnormalities (pup 7 of litter 58) were collected and fixed in 10% buffered formalin. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk. If possible, defects or cause of death were evaluated.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-toone t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Kidneys in high and intermediate dose males

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

Pale discolouration of the kidneys (bilateral) was noted for one male at 300 mg/kg and five males at 1000 mg/kg.
The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included pelvic dilation of the kidneys, yellowish foci or nodules on the epididymides, dark red foci on the lungs or thymus, reddish discolouration of the mandibular lymph nodes, hardening and dark red discolouration of the papillary process of the liver, alopecia, and early resorption in the uterus.

There were no test item-related microscopic findings in the reproductive organs examined.
The kidneys of male rats, which were discolored pale at necropsy, showed an increase in hyaline droplets: Hyaline droplet accumulation was recorded in 1/1 male rat at 300 mg/kg/day at a moderate degree, combined with minimal tubular basophilia and slight granular casts. At 1000 mg/kg/day hyaline droplet accumulation was recorded in 6/6 male rats (2: slight, 4: moderate), in all 6 males combined with tubular basophilia (3: minimal, 2: slight, 1 moderate) and in 3/6 males combined with moderate granular casts. Hyaline droplet accumulation was also recorded in 1/2 males (minimal) of the control group 1, and in 1/1 male at 100 mg/kg/day (slight).
There were two pairs at 300 mg/kg/day (male 25/female 65, male 30/female 70) and one pair at 1000 mg/kg/day (male 39/female 79) that failed to sire or deliver healthy pups. No cause for the failure to sire or deliver healthy offspring in these animals could be established from the sections examined.
In addition female 80 (1000 mg/kg/day) showed a total litter loss on Day 1 of lactation (all pups were dead at first litter check) and was therefore sacrificed. Necrotic remnants were present in the uterus of this animal. Necrotic remnants were present in the uterus of this animal and probably the cause of the death of this litter.
All remaining findings (including the spermatogenic staging profiles) were within the normal range of background pathology.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproduction and developmental toxicity was observed up to the highest dose level tested (1000 mg/kg)

Target system / organ toxicity (P0)

Key result

Critical effects observed:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

Gestation
Gestation index and duration of gestation were unaffected by treatment.
The occurrence of a total litter loss for female no. 80 at 1000 mg/kg (all 13 pups were dead at first litter check) was not considered toxicologically relevant at this single incidence.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females.
Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Early postnatal pup development
Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.
The statistically significantly decreased number of living pups at first litter check at 300 mg/kg was not considered toxicologically relevant as all values were within normal limits and it showed no dose related trend. Moreover, when including the thirteen pups of litter no. 80, it resulted in a mean number of 10.7 pups per litter at 1000 mg/kg.

Mortality
All thirteen pups of one litter (no. 80) at 1000 mg/kg were found dead at first litter check. At this single occurrence, it was not considered toxicologically relevant.
At 100 mg/kg, one pup (litter 54) was killed in extremis on Day 4 of lactation due to a large wound caused by the dam. Another pup at 100 mg/kg and three pups at 300 mg/kg were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Observations
Incidental findings for pups consisted of scabbing of several body parts, blue discolouration of the back, or missing tail. The pup that was killed in extremis showed a large wound on Day 4 of lactation.
The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Body weights
Body weights of pups were considered to have been unaffected by treatment.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproduction and developmental toxicity was observed up to the highest dose level tested (1000 mg/kg)

Target system / organ toxicity (F1)

Key result

Critical effects observed:

not specified

Overall reproductive toxicity

Key result

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.

Executive summary:

The Reproduction/developmental toxicity of the substance has been assessed by means of the screening test by oral gavage according to the OECD 421, Reproduction/Developmental Toxicity test guideline.The dose levels for this reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg.

The test substance, formulated in corn oil, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination.

Females were exposed for 37-54 days, i.e. during 2 weeks prior to mating, during mating, during postcoitum, and during at least 4 days of lactation.

Accuracy, homogeneity and stability of formulations were demonstrated by analyses.

Parental results:

At 300 and 1000 mg/kg, toxicity consisted of kidney findings for males.

The pale discoloured kidneys recorded at necropsy for one male at 300 mg/kg and five males at 1000 mg/kg showed slight-moderate hyaline droplet accumulation, which was the microscopic correlate to this finding. These hyaline droplets were considered to represent alpha2μglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation leading ultimately to proximal cortical tubule cell injury, the formation of granular casts and increased cell turnover as manifest by tubular basophilia (Ref. 5). This protein is not present in female rats nor in higher mammals, including man. Therefore, these findings are not predictive for man and their occurrence has no relevance in human safety assessment.

Reproductive and developmental results:

No reproduction and developmental toxicity was observed up to the highest dose level tested (1000 mg/kg)