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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test”
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): 1,2,3-Propanetriol, homopolymer, diisooctadecanoate
- Physical state: yellowish/clear viscous liquid
- Analytical purity: 100% (reaction mixture)
- Batch No.: CD11110003
- Expiration date of the lot/batch: 21-Apr-2013
- Storage condition of test material: room temperature (15 - 25 °C)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 weeks
- Weight at study initiation: 260 - 390 g (males), 175 - 240 g (females)
- Housing: in groups of three to five in Makrolon type-4 cages with wire mesh tops during acclimatisation and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding with paper enrichment. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet (ad libitum): pelleted standard Harlan Teklad 2018C rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland)
- Water (ad libitum): community tap-water
- Acclimation period: at least seven days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): Based on an observation on 01-Feb-2013, the automatic light cycle of 12h on / 12 h off was not functional within a block of rooms including room numbers 015A and 011B. Due to the breeding performance from November 2012 onwards during this and other studies conducted in rooms which belong to the affected block, it was considered probable that the animals hab been maintained under constant lightning throughout the study period for the first delivery animals. From 01-Feb-2013 onwards, the automatic light cycle was restored.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: daily. The test substance was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.
Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 4 mL/kg bw (dose volume)
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation was observed
- Proof of pregnancy: copulation plug or sperm in vaginal smear referred to day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no, if mating was not recorded during the additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
On the first dose formulation day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 1 g of each test item concentration were taken from the middle to confirm the stability (4 hours). Towards the end of the study, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and stored at -20 ± 5 °C until analysis.
The samples were analyzed by derivatisation with MSTFA and analysis by GC-FID. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.
Duplicates were taken of all samples and stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were not discarded without written consent from the study director.
Duration of treatment / exposure:
Males: Minimum 28 days
Females: Approximately 6 weeks
Frequency of treatment:
once daily (within four hours)
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
at least 12 (see any other information on materials and methods)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: - Dose selection rationale: Based on single dose toxicity data of the test compound and repeated dose toxicity data of chemically similar compounds, the results of a 14-day range-finding study the dose levels of 100, 300, and 1000 mg/kg bw per day were selected for the present study.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration of the test item and weekly thereafter (in the gestation period on day 0, 6, 13 and 20 post coitum).
- detailed clinical observations were performed outside the home cage in a standard arena and the animals were observed for the following changes in: skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies or bizarre behavior were also reported.

BODY WEIGHT: Yes
- Time schedule for examinations: daily from treatment start to day of necropsy.

FOOD CONSUMPTION: Yes
- Males: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; after pairing period weekly.
- Females: Pre-pairing period days 1 - 4, 4 - 8, 8 - 11 and 11 - 14; gestation days 0 – 7, 7 14 and 14 – 21 and days 1 - 4 of the lactation
No food consumption was recorded during the pairing period.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
testis weight, epididymis weight, daily sperm production, seminal vesicles, stages of spermatogenesis
Litter observations:
STANDARDISATION OF LITTERS
The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed
individually (without identification) on days 1 and 4 post partum.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other:]
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
The following organs were trimmed from any adherent tissue, as appropriate, and their wet weight was taken: adrenal glands (weighed as pairs), brain, epididymides, heart, kidneys (weighed as pairs), liver, ovaries (weighed as pairs), prostate, seminal vesicles (inclusive coagulating gland), spleen, testes, thymus, thyroid (after fixation), uterus (including cervix)
HISTOPATHOLOGY: Yes
The following tissues from all parental animals were preserved in neutral phosphate buffered 4% formaldehyde solution if not otherwise described: adrenals, brain, bone marrow (femur), epididymides (in Bouin’s fixative), gross lesions, heart, kidneys, liver, lymph nodes (axillary and mesenteric), ovaries (with oviduct), peripheral nerve (sciatic), prostate, seminal vesicles with coagulating gland, small and large intestines (incl. Peyer’spatches; duodenum, jejunum, ileum, colon, caecum, rectum), spinal chord (cervical, thoracic, lumbar), spleen, stomach (forestomach and glandular stomach), testes (in Bouin’s fixative), thymus, thyroids, and parathyroids if possible, trachea and lungs (preserved by inflation with fixative and then immersion), uterus (with vagina), urinary bladder
The following tissues were only examined by histopathology in case of macroscopic findings indicative of potential toxicity: aorta, esophagus, eyes with optic nerve and harderian gland, femur with knee joint, lacrimal gland, larynx, mammary gland (male and female), nasal cavity, pancreas, pharynx, pituitary gland, salivary glands – mandibular, sublingual, skeletal muscle, sternum with bone marrow.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at day 4 post partum.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
All parent animals and pups were examined macroscopically for any structural changes at the
scheduled necropsy.
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables were assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test will be applied if the variables could be dichotomized without loss of information
Reproductive indices:
Percentage mated = (Females mated/Females paired)*100
Fertility index = (Females achieving a pregnancy/Females paired) *100
Conception rate = (Females achieving a pregnancy/Females mated)*100
Gestation index = (Number of females with living pups / Females pregnant) * 100
Offspring viability indices:
Birth index = (pups born alive/number of implantations)*100
Viability index = (pups alive before cullig on day 4 post partum/pups born alive)*100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see details on results
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: see details on results

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
see details on results

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
At a dose level of 1000mg/kg bw/day, one male (no. 171, third animal delivery) died spontaneously on day 1 of the after pairing period. No clinical signs were observed and a normal body weight gain was recorded until day 13 of the pairing period. One day before it died, it showed a body weight loss of 3%. During necropsy, a dark red discolored thymus was recorded.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males:
Mean body weight gain was statistically significantly lower at 1000mg/kg bw/day during pre-pairing period, resulting in slightly reduced mean body weights (-3% at the end of this period compared to the controls). This was a result of the minor body weight loss (1%) between days 1 and 2. During the pairing period, body weight gain at the high dose level was similar to the control value. Due to this fact, teh reduction in mean body weight gain ans body weights were considered to be not adverse. The overall differences in mean body weight gain at the dose levesl of 0, 100, 300 and 1000mg/kg bw/day were: +13%, +12%, +11% and +11% during the pre-pairing period, +5%, +5%, +3% and +4% during the pairing period and +7%, +9%, +7% and +8% during the after pairing period (percentages refer to the body weight gain within the period).
At 1000mg/kg bw/day, mean food consumption was statistically significantly reduced during the first 11 days of treatment in males of the first and second delivery (-9,3% on day 11 of prepairing when compared with controls). Thereafter, the food consumption remained slightly lower than in controls, but without reaching statistical signifincance. Fod consumption was also lower in males of the third delivery, but not to a statistically significant degree. Since this reduction in food consumption occured only transiently, the reduction was considered to be not adverse.

Females: No effect of the test item on mean body weights and mean body weight gain was noted for females at any dose level. Towards the end of the gestation period, body weights and body weight gain were reduced in females at 1000mg/lg bw/day (statistically significantly different body weight gains drom day 17 post coitum onwards). These differences were considered to be a result of the increased post implantation loss and low number of pups, which were noted for dams at this dose level. The overall differences in mean body weight gain at the dose levels of 0, 100, 300 and 1000mg/kg bw/day were: +8%, +9%, +8% and +7% during the pre-pairing , +63%, +57%, +57% and +51% during the gestation period and +4%, +7%, +4% and +3% during days 1 to 4 of the lactation period (percentage refer to the body weight gain within the period.
There were no effects on mean food consumption at any dose level and in any study phase.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The fertility index and conception rate were low across all groups in females of the first animal delivery only. This poor mating performance was considered to be due to a disturbance of the light / dark cycle during the conduct of this study and not test item-related. In females at 1000 mg/kg bw/day, the mean duration of gestation was slightly higher than in controls (22.1 days versus 21.3 days in the controls for first and second animal delivery, 21.7 days versus 21.3 days in the controls for third animal delivery) and not in the range or borderline to the historical control data (21.2 to 21.7 days). Also, the post-implantation loss was increased (16.2% versus 6.6% in controls for first and second animal delivery, 14.2% versus 3.3% in
controls for third animal delivery), which was borderline regarding the historical control data (3.3% - 14.4% as a percentage of total implantations).
Due to the higher post-implantation loss, mean litter size at first check was reduced at the high dose level.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Treatment with the test item at 1000mg/kg bw/day resulted in statistically significantly higher absolute and relative liver and kidney weights in animals of bith genders, which was confirmed in animals of the third delivery except for kidney weights in females. Since there was no evidence for an impairment of organ function by clinical pathology and histopathology, these findings were not considered to be adverse. Additionally, in females, absolute and relative heart weights were also increased. This finding was without histopathological correlate, and was therefore considered to be incidental. There were no effects of treatment at 100 and 300mg/kg bw/day.

GROSS PATHOLOGY (PARENTAL ANIMALS)
All findings were typical of this strain and age of rat and were considered to be incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment-related findings. In particular, qualitative examination of the stages of spermatogenesis in the testis did not reveal any treatment-related abnormalities in the integrity of various cell types present within the different stages of the sperm cylce. No treatment-related microscopic abnormalities were observed in the evaluation of the ovarian follicles and corpora lutea of the ovaries or the evaluation of the uterus. All findings were typical of this strain and age of rat and were considered to be incidental.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Dose descriptor:
NOEL
Remarks:
sytemic
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
see details on results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see details on results
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):
see details on results
Histopathological findings:
not examined

Details on results (F1)

VIABILITY AND CLINICAL SIGNS (OFFSPRING)
No test item-related findings were noted in pups during first litter check and during lactation at any dose level.
Three control pups from the litter no. 129 showed no milk in the stomach on the first day post partum. Those three pups were then missing at litter check on day 2 post partum, along with a further female pup. One pup of this litter had already been found dead at first litter check. The
remaining 10 pups from this litter survived until their necropsy on day 4 post partum.
At 1000 mg/kg bw/day, one pup showed a black discolored exophthalmos on the first day post partum. This animal was free from clinical signs from day 2 post partum onwards and survived until necropsy on day 4. A further pup showed a wound on the head on days 3 and 4 post partum.

BODY WEIGHT (OFFSPRING)
Mean pup weights on day 1 and day 4 post partum were unaffected by treatment with the test item.
On day 1 post partum mean pup weights were 5.7, 6.1, 6.0 and 5.9 g in order of ascending dose level. Also mean body weight gain was similar in all groups on day 4 post partum and no effects on mean body weights were recorded.

GROSS PATHOLOGY (OFFSPRING)
No test item-related findings were noted at macroscopic examination of F1 pups.
One male pup treated at 100 mg/kg bw/day had a missing right testicle at planned necropsy and two pups treated at 1000 mg/kg bw/day showed sores on the skin. These isolated findings were incidental and unrelated to treatment.

OTHER FINDINGS (OFFSPRING)
Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item.
The proportion of males at first litter check was 56, 53, 50 and 51%, in order of ascending dose level.

Effect levels (F1)

Dose descriptor:
NOEL
Remarks:
reproduction and development
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on these results, the NOAEL (No Observed Adverse Effect Level) for general toxicity was considered to be 1000 mg/kg body weight/day. The NOEL (No Observed Effect Level) for general toxicity and for reproduction and development was established at 300 mg/kg bw/day.
Executive summary:

A study investigating the toxicological effects of the test item 1,2,3 -Propanetriol, homopolymer, diidoctadecanoate to rats according to OECD guideline 422 resulting from repeated oral-gavage administration is performed. 1,2,3 -Propanetriol, homopolymer, diidoctadecanoate was administered in corn oil as vehicle at dosages of 100, 300, 1000 mg/kg bw/day, and control received the vehicle control only.

To meet the guideline requirements concerning number of pregnant females per group, male and female rats were added in a second delivery for breeding due to reduced fertility observed in all groups of the first delivery. It is believed, that a disturbance of the light / dark cycle was the reason for the reduced mating rate of the females of the first delivery. The additional animals ran through the whole study as the animals from the first delivery with the exception that the males went to necropsy on day 24 of the after pairing period and not on day 16 of the pairing period as the males of the first delivery (logistical reasons). Therefore, the test item was administered to male rats for up to 28 days (first delivery) and up to 41 days (second delivery) and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

Because an impact of the light/dark cycle disturbance during the study on these results (a prolonged duration of gestation and an increased post-implantation loss was observed at the high dose) could not be excluded, the study was repeated with a third animal delivery of control and high-dose (1000 mg/kg bw/day) groups under proper light conditions. The test item was administered

to 12 male rats per group for up to 33 days and to 12 female rats per group for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

All animals survived until scheduled necropsy. There were no treatment-related clinical signs or effects on fuctional observational battery or locomotor activity. No test-titem related findings were noted during the clinical laboratory investigations.

Liver and kidney weights were slightly increased at 1000 mg/kg bw/day in both sexes of the first and second delivery, which was confirmed in animals of the third delivery except for kidney weights in females. These changes were considered compound-related, but not adverse, since there was no evidence for an impairment of organ function by clinical pathology and histopathology. There were no treatment-related necropsy or microscopic findings.

Overall at 1000 mg/kg bw/day, food consumption and body weight gain was transiently reduced in males at the beginning of the treatment period which was considered to be not adverse. The mean duration of gestation was slightly higher than in controls and the post-implantation loss was increased (up to 16.2% versus 6.6% in controls), which was borderline regarding the historical control data (3.3% - 14.4% as a percentage of total implantations). At 1000 mg/kg bw/d some changes in clinical biochemistry were noted in females. Although these effects were not dose-dependant and within the historical control range, a test item-related effect can´t be excluded. Therefore it is possible that the higher implantation losses and reduced litter size at 1000 mg/kg bw/d were induced by maternal toxicity, even if the observed effects are not regarded as adverse to the parental animals. According to the applicant, the borderline effects on post-implantation losses and litter size, should not be regarded a seperat toxicity to reproduction.

Based on these results, the NOAEL (No Observed Adverse Effect Level) for general parental toxicity was considered to be 1000 mg/kg body weight/day. The NOEL (No Observed Effect Level) for general parental toxicity and for reproduction and development was established at 300 mg/kg bw/day.