Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: Combined repeated dose and reproduction/developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Reliability: Guideline study, well-performed and well documented. read-across justification for read.across: the grouped substances differ only that the registraton substance is a weak acid and the read-across substance is the corresponding weak base. When dissolved in water the read-across supporting substance will be converted to the registration substance.
Reason / purpose:
reference to same study
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 (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 338 to 369 g (males) and 212 to 242 g (females)
- Identification: Parent animals had cage cards and individual animal numbers (ear tattoos) and pups were individually tattooed with Indian ink on day 1 post partum.
- Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Acclimatization: 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occured, which is considered not to have any influence on the study. These data were not reported but will be retained in the raw data. There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: In groups of five in Makrolon type-4 cages with wire mesh tops up to the day of randomization and afterwards individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands) batch/lot nos. 02105111001, 02105111201, 6960C.CS-100099 and 72). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2018C (batch no. 80/11) rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum. Analyses for contaminants were performed - Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles. A bacteriological assay, chemical and contaminant analyses of representative samples were performed.

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
DOSE FORMULATIONS

The dose formulations were prepared weekly using the test item as supplied by the Sponsor.

(Pentapropylensuccinimido)-hexanoic acid, sodium and triethanolamine salts was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogene¬ous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration and divided into daily aliquots.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS

Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers.

Based upon the results of stability analyses performed within the Harlan Laboratories study D28366, dose formulations were stable for at least 8 days.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Target Dose Levels: 0 mg/kg/day (Group 1, control group), 40 mg/kg/day (Group 2), 200 mg/kg/day (Group 3) and 1000 mg/kg/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories study D28366, using dose levels of 0, 100, 300 and 1000 mg/kg/day.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL/day (Group 1), 4 mg/mL/day (Group 2), 20 mg/mL/day (Group 3) and 100 mg/mL/day (Group 4)
Details on mating procedure:
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum.

If a female did not mate during the 14-day pairing period, a second pairing of this female with a male in the same group, which had already mated successfully, was considered.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm the stability (8 days, stored at room temperature). During the last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to the analytical department (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to an UV detector following an analytical procedure provided by the Sponsor and adapted at Harlan Laboratories. The test item was used as the analytical standard.

Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland.

RESULTS

The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. All calibration points used met the acceptance limit of ±20% variation from the calibration curve derived by linear regression analysis. The regression coefficients (R2) calculated were found to be better than 0.99.

The test item peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no peak appeared at the retention time of test item and, therefore, the absence of the test item in the vehicle control samples (highly purified water) was confirmed.

The application formulations investigated during the study were found to comprise test item in the range of 91.1% to 97.3% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of test item in the preparations was approved because single results found did not deviate more than 0.6% (acceptance criterion: ≤15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item and highly purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was proved.
Duration of treatment / exposure:
- Males: Minimum 4 weeks
- Females: Approximately 7 weeks
Frequency of treatment:
Once daily
Details on study schedule:
MALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment End: On day before sacrifice
- Blood Sampling: On the day of necropsy
- Necropsy: After treatment for 28 days, when no longer needed for assessment of reproductive effects

FEMALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment End: On day 4 post partum
- Blood Sampling: Day 5 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)
Remarks:
Doses / Concentrations:
0, 40, 200, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
The purpose of this study was to generate preliminary information concerning the effects of (Pentapropylensuccinimido)-hexanoic acid, sodium and triethanolamine salts on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

(Pentapropylensuccinimido)-hexanoic acid, sodium and triethanolamine salts was administered to male rats for 28 days 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.
Positive control:
Not required
Parental animals: Observations and examinations:
VIABILITY/MORTALITY

Twice daily

CLINICAL SIGNS

Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION

Males: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 of the pre-pairing period and weekly during the after pairing period.
Females: on days 1 - 4, 4 - 8, 8 - 11 and 11 - 14 of the pre-pairing period; on days 0 - 7, 7 - 14 and 14 - 21 of the gestation period and on days 1 - 4 of the lactation period.

No food consumption was recorded during the pairing period.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy.

DETAILED CLINICAL OBSERVATIONS

Detailed clinical observations were performed in all animals outside the home cage. In males, it was performed once prior to the first administration of the test item and weekly thereafter. In females, it was performed once prior to the first test item administration, weekly during the pre-pairing and pairing periods and on days 0, 6, 13 and 20 of the gestation period.

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.


FUNCTIONAL OBSERVATION BATTERY

At one time during the study (males shortly before the scheduled sacrifice and females on day 3 or 4 post partum), relevant parameters were observed in five P generation males and five P generation females from each group. This FOB assessment was conducted following the daily dose administration. Animals were observed for the following:

- Cage-side observations: faeces-balls, urine and posture as well as resistance to removal.
- Hand-held observations: muscle tone, constituation, skin, pupile size, palpebral closure, lacrimation, salivation, reaction to handling and general abnormalities.
- Open field observations: level of ambulatory activity including rearing (one minute evaluation), unusual body movements (e.g. spasms, convulsions), gait evaluation, behavior, hair coat, respiration, quantity of faeces-balls and urine.
- Reflexes: blinking, palpebral closure, pinna reflex, extensor thrust response, paw pinch, responsiveness to sharp noise, righting reflex and hearing ability (Preyer’s reflex).
- Measurements / Counts: hind limb / fore limb grip strength, landing foot splay, rectal temperature.

Additionally, locomotor activity was measured quantitatively for the same animals. Activity was measured with an Activity Monitor AMS-0151 (FMI, Germany). Activity of the animals (based on beam count) was recorded for 6-minute intervals over a period of 30 minutes. These data and the total activity over 30 minutes were reported.

CLINICAL LABORATORY INVESTIGATIONS

Blood samples were obtained on the day of the scheduled necropsy from 5 males from each group. Blood samples from 5 lactating females from each group were obtained on day 5 post partum. Blood samples were drawn sublingually from all animals under light isoflurane anesthesia. The animals were fasted for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms.

The following hematology parameters were determined:
- Erythrocyte count
- Hemoglobin
- Hematocrit
- Mean corpuscular volume
- Red cell volume distribution width
- Mean corpuscular hemoglobin
- Mean corpuscular hemoglobin concentration
- Hemoglobin concentration distribution width
- Leukocyte count, total
- Differential leukocyte count
- Platelet count
- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

The following clinical biochemistry parameters were determined:
- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Bile acids
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
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 0 (if possible), 1 and 4 post partum.
Postmortem examinations (parental animals):
TERMINATION AND NECROPSY

Males were sacrificed after treatment of at least 28 days, when no longer needed for the assessment of reproductive effects. Pups were sacrificed on day 4 post partum. Dams were sacrificed on day 5 post partum. If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

At the scheduled sacrifice, all parent animals were sacrificed by an injection of sodium pentobarbital and exsanguinated. All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death. Special attention was directed at the organs of the reproductive system.

Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

ORGAN WEIGHTS

At the scheduled sacrifice, testes and epididymides from all parental males were weighed separately. In addition, from 5 males and 5 females killed at the end of the study which were selected from each group, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken:
- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Thymus
- Spleen

TISSUE PRESERVATION

The following tissues from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Prostate
- Seminal vesicles with coagulating gland
- Testes (in Bouin’s fixative)
- Epididymides (in Bouin’s fixative)*

The following tissues from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries

In addition, from the 5 males and 5 females per group selected for organ weights and from all animals found dead or killed in extremis, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Gross lesions
- Brain (representative regions including cerebrum, cerebellum and pons)
- Spinal chord
- Small and large intestines (incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids, and parathyroids if possible
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Uterus (with vagina)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Bone marrow

HISTOTECHNIQUE

All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis was stained by PAS-hematoxylin. Special stains were used at the discretion of the study pathologist.

HISTOPATHOLOGY

Slides of all organs and tissues listed collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions and to all animals which died spontaneously or had to be terminated in extremis.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

When test item-related morphologic changes were detected in organs of any high-dose animal, those same organs from the mid- and low-dose group were examined to establish a no-effect level, if possible.

Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of all infertile males was made, if necessary.

A histopathology peer review was performed. A histopathology phase report was provided by the principal investigator and was included in the report.
Postmortem examinations (offspring):
Pups were sacrificed on day 4 post partum. All animals were sacrificed by an injection of sodium pentobarbital and subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. Pups dead during the study, except those excessively cannibalized, were examined macroscopically.
Statistics:
The following statistical methods were used to analyze food consumption, body weights, locomotor activity, grip strength, body temperature, hematology, clinical biochemistry and reproduction data:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be 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 was applied if the variables could be dichotomized without loss of information.
Reproductive indices:
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size, pup sex ratios and viability indices.
For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis.
Offspring viability indices:
From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pups sex ratio and viability index.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation and bedding in mouth in several (group 2) or all animals (groups 3 and 4). Decreased activity in 2 females (group 3) or 4 males and 4 females (group 4). Furthermore, rufffled fur and yellow discolored faeces in all group 4 animals
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduction in food consumption in all animals of all dose groups. Reduction in groups 3 and 4 was considered adverse. Reduction in group 2 was considered not to be adverse. Reduction in body weight and body weight gain in groups 3 and 4 considered adverse.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduction in food consumption in all animals of all dose groups. Reduction in groups 3 and 4 was considered adverse. Reduction in group 2 was considered not to be adverse. Reduction in body weight and body weight gain in groups 3 and 4 considered adverse.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Effects on liver weights (males and females) and spleen weights (males) in group 4. Also, reduces testis and epididimides weights. Possible effect of treatment.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(Effects in group 4 consisting of central to diffuse hypertrophy (liver), increased incidence of hyaline droplets (kidney epthelium), squamous hyperplasia (foresomach - also in few group 2 and 3 animals) and follicular celly hypertrophy (thyroid gland))
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Higher number of non pregnant females in group 4. Increased post-implantation loss, reduced birth and viability indexes, lower litter size in groups 2 to 4.
1. IN-LIFE DATA

VIABILITY/MORTALITY

All animals survived until the scheduled necropsy.

DAILY CLINICAL SIGNS OR OBSERVATIONS

Treatment with the test item at the dose level of 1000 mg/kg bw/day caused salivation, bedding in mouth and ruffled fur in all males and females. Decreased activity was noted in all males and four females. In addition, yellow discolored faeces were noted in all males and females at the high dose level; this finding was a result of staining properties of the test item.

Treatment with the test item at the dose level of 200 mg/kg bw/day caused salivation and bedding in mouth in all males and females. Decreased activity was noted in two females at this dose level.

At the dose level of 40 mg/kg bw/day, salivation and bedding in mouth were noted in several males and females for one or two days during the pre-pairing period.

No further test item-related clinical signs or observations were noted in males or females at any dose level.

FINDINGS AT DETAILED WEEKLY CLINICAL OBSERVATIONS

During weekly detailed clinical observations, ruffled fur and decreased activity were confirmed in males and females at the dose level of 1000 mg/kg bw/day.

No further test item-related findings were noted in males or females at any dose level.

FUNCTIONAL OBSERVATIONAL BATTERY

At the dose level of 1000 mg/kg bw/day, ruffled fur was confirmed in all males and females. Further findings at this dose level, which were considered to be test item-related, were: decreased rearings (in two males and one female), increased faeces balls (in three males and one female) and reduced body temperature in both sexes. Reduction of body temperature was statistically significant; mean body temperature at the high dose level was in males 37.6 °C, compared to 38.3 °C in the control group and in females 37.7 °C compared to 38.7 °C in the control group.

At the dose level of 200 mg/kg bw/day, increased faeces balls were noted in one female.

No further test item-related findings were noted during functional observational battery in males or females at any dose level.

LOCOMOTOR ACTIVITY

At the dose levels of 1000 and 200 mg/kg bw/day, reduced locomotor activity was noted in males and females. In males, reduction of locomotor activity was statistically significant; mean beam counts per minute were 425 and 273 at the high- and mid-dose levels, respectively, whereas in the control group 1294 counts per minute were recorded. In females, differences to the control values were not statistically significant; mean beam counts per minute were 577 and 763 at the high- and mid-dose levels, respectively, and 974 counts per minute in the control group.

At the dose level of 40 mg/kg bw/day, locomotor activities of males and females were similar to the respective control values.

FOOD CONSUMPTION OF MALES

Food consumption was reduced in all dose groups.

At the dose level of 1000 mg/kg bw/day, mean food consumption was 17.9 g per animal per day compared to 26.1 g in the control group. If expressed as a percentage of the control value, the difference in mean food consumption was 31.4%. Reduction in food consumption was statistically significant during the entire pre-pairing period.

At the dose level of 200 mg/kg bw/day, mean food consumption was 23.4 g per animal per day, which corresponded to a difference of 10.3% if compared to the control value. Reduction in food consumption was statistically significant on days 1 - 4 and 11 - 14 of the pre-pairing period.

At the dose level of 40 mg/kg bw/day, mean food consumption was 25.4 g per animal per day, which corresponded to the difference of 2.7% if compared to the control value. Reduction in food consumption was statistically significant on days 11 - 14 of the pre-pairing period.

FOOD CONSUMPTION OF FEMALES

Food consumption was reduced in all dose groups.

At the dose level of 1000 mg/kg bw/day, mean values of food consumption during the pre-pairing, gestation and lactation periods were 13.5 g, 19.0 g and 15.9 g, whereas control values were 19.5 g, 26.1 g and 30.3 g per animal per day, respectively. If expressed as a percentage of the control value, the differences in mean food consumption were -30.8%, -27.2% and -47.5% during the pre-pairing, gestation and lactation periods, respectively. Reduction in food consumption was statistically significant during the entire pre-pairing, gestation and lactation periods.

At the dose level of 200 mg/kg bw/day, mean values of food consumption during the pre-pairing, gestation and lactation periods were respectively 17.2 g, 22.4 g and 20.0 g per animal per day, which corresponded to the differences of -11.8%, -14.2% and -34.0% if compared to the respective control values. Reduction in food consumption was statistically significant on days 1 - 8 and 11 - 14 of the pre-pairing period and entire gestation and lactation periods.

At the dose level of 40 mg/kg bw/day, mean values of food consumption during the pre-pairing, gestation and lactation periods were respectively 18.8 g, 23.7 g and 27.6 g per animal per day, which corresponded to the differences of -3.6%, -9.2% and -8.9% if compared to the respective control values. Reduction in food consumption was statistically significant on days 0 - 14 of the gestation period.

BODY WEIGHTS OF MALES

Treatment with the test item caused reduction in body weights and transient reduction in body weight gain at the dose levels of 1000 and 200 mg/kg bw/day.

At the dose level of 1000 mg/kg bw/day, body weights were statistically significantly reduced starting from day 4 of the pre-pairing period until the completion of the study.
Slight decrease of body weights (by 2%) was noted at this dose level between day 1 and 4 of the pre-pairing period. Afterwards, body weight gain was stable although until the completion of the pre-pairing period remained lower than the control values. During the pairing period body weight gain increased and was higher than the control values. Reduction in body weight gain was statistically significant starting from day 2 until the completion of the pre-pairing period, increase in body weight gain was statistically significant on day 3 and from day 5 until the completion of the pairing period.

Mean differences in body weight gain were -1% during the pre-pairing period and +9% during the pairing period compared to the respective values of +11% and +6% in the control group.

At the dose level of 200 mg/kg bw/day, body weights were statistically significantly reduced starting from day 12 of the pre-pairing period until day 9 of the pairing period.
Slight decrease of body weights (by 1%) was noted at this dose level on day 2 of the pre-pairing period. Afterwards, body weight gain increased although until the completion of the pre-pairing period remained lower than the control values. Reduction in body weight gain was statistically significant starting from day 11 until the completion of the pre-pairing period. During pairing period, body weight gain was similar to the control values.

Mean differences in body weight gain were +7% during the pre-pairing period and +7% during the pairing period.

At the dose level of 40 mg/kg bw/day, body weights and body weight gain were considered not to be affected by the treatment. Mean differences in body weight gain were +8% during the pre-pairing period and +8% during the pairing period.
On day 12 of the pre-pairing period, body weight gain was statistically significantly lower when compared to the control value. Because during the remaining study period body weight gain and body weights were similar to the respective control values, the isolated difference in body weight gain on day 12 of the pre-pairing period was considered to be incidental.

BODY WEIGHTS OF FEMALES

Treatment with the test item caused reduction in body weights at the dose levels of 1000 and 200 mg/kg bw/day and reversible reduction of body weight gain at the dose level of 1000, 200 and 40 mg/kg bw/day.

At the dose level of 1000 mg/kg bw/day, body weights were statistically significantly reduced starting from day 4 of the pre-pairing period until the completion of the study.

Slight decrease of body weights (by 2%) was noted at this dose level between day 1 and 4 of the pre-pairing period. Afterwards, body weight gain increased slightly although until the completion of the pre-pairing period remained lower than the control values. During the gestation and lactation periods body weight gain remained lower than the control values. Reduction in body weight gain was statistically significant starting from day 3 until the completion of the pre-pairing period and during the entire gestation period.

Mean differences in body weight gain were +2% during the pre-pairing period, +33% during the gestation period and ±0% during the lactation period compared to the respective values of +9%, +57% and +5% in the control group.

At the dose level of 200 mg/kg bw/day, body weights were similar or slightly, not statistically significantly lower than the respective control values during the most of the study period. Statistically significantly lower body weights were noted on days 4 and 5 of the lactation period.

Slight decrease of body weights (by 1%) was noted at this dose level on day 2 of the pre-pairing period. Afterwards, body weight gain increased slightly but until the completion of the gestation period remained lower than the control values. During lactation period, body weight gain was similar to the control values. Reduction in body weight gain was statistically significant on days 2, 11 and 14 of the pre-pairing period and on days 5, 11, 13 and 14 of the gestation period.
Mean differences in body weight gain were +6% during the pre-pairing period, +49% during the gestation period and +4% during the lactation period.

At the dose level of 40 mg/kg bw/day, body weight gain was occasionally lower if compared to the control values; slight decrease of body weights (by 1%) was noted on day 2 of the pre-pairing period. On day 14 of the pre-pairing period and day 3 of the gestation period body weight gain was statistically significantly lower than the control values. During the remaining study period body weight gain at the low-dose level was similar or slightly lower then the respective control values. No significant differences in body weights were noted at this dose level. Change in body weight gain was considered to be test item-related, not adverse effect.

Mean differences in body weights were +6% during the pre-pairing period, +55% during the gestation period and +6% during the lactation period.

2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

At the dose level of 1000 mg/kg bw/day, statistically significantly higher platelet count and statistically significantly higher relative prothrombin time (PT) were noted in males. Mean platelet count at this dose level was 1318 x 109/L compared to 1004 x 109/L in the control group, mean relative protrombin time was 0.90 compared to 0.82 in the control group. These changes were considered to be test item-related. No further effects on hematology parameters were noted in males at any dose level.

No effects on hematology parameters, which were considered to be test item-related, were noted in females at any dose level. Incidentally, statistically significantly higher total leukocyte count (WBC) was noted at the low-dose level. Because no changes of this parameter were noted at mid- or high-dose levels, the higher WBC was not test item-related. No further changes of hematology parameters were noted in females at any dose level.

CLINICAL BIOCHEMISTRY

At the dose level of 1000 mg/kg bw/day, increased level of potassium was noted in males. Mean concentration was 4.60 mmol/L at the high-dose level and 4.00 mmol/L in the control group. Concentration of potassium at the high dose level was within the historical control background containing values from 3.33 to 6.30 mmol/L. However, a slight but dose dependent changes of potassium concentration was observed in males across all dose groups and therefore this effect was considered to be possibly test item-related.

Remaining statistically significant changes in males were noted at the dose level of 200 mg/kg bw/day; lower concentration of bilirubin (BILI-T), higher concentration of phosphorus and lower concentration of protein and albumin. None of these changes occurred at the dose level of 1000 mg/kg bw/day and therefore they were considered not to be related to the treatment with the test item.

In females, at the dose level of 1000 mg/kg bw/day, increased concentration of total protein and increased concentration of albumin were noted. Mean concentrations were 72.11 g/L at the high dose level versus 65.35 g/L in the control group for total protein and 47.27 g/L at the high dose level versus 41.98 g/L in the control group for albumin. Values at the high dose level exceeded the historical control background containing values of total protein from 61.16 to 71.25 g/L and values of albumin from 37.65 to 44.47 g/L. For this reason, test item-related changes of total protein and albumin concentrations should be taken into consideration.Remaining changes of biochemical parameters in females were considered not to be related to the treatment with the test item. At the dose level of 40 mg/kg bw/day, statistically significantly higher activity of alanine aminotransferase (ALAT) was noted but without increase of the activity at the dose level of 200 and 1000 mg/kg bw/day. At the dose level of 1000 mg/kg bw/day, a higher concentration of cholesterol and lower concentration of phosphorus were noted. These changes were not dose dependent and no similar observations were done in males.

No further changes in biochemical parameters were noted at any dose level.

3. TERMINAL FINDINGS

ORGAN WEIGHTS

In males treatment with the test item at the dose level of 1000 mg/kg bw/day caused increase in liver and spleen weights. Mean liver weigh was 13.39 g versus 9.83 g in the control group. Mean spleen weight was 0.86 g versus 0.70 g in the control group. Increase in absolute weights and increase in weights of these organs relative to body weight and to brain weight were statistically significant.

Further, at the dose level of 1000 mg/kg bw/day, statistically significantly reduced absolute testis and epididymides weighs as well as statistically significantly reduced epididymides weights relative to brain weights were noted. Mean testis weights (left/right) were 1.88/1.84 g versus 2.07/2.04 g in the control group. Mean epididymides weights were 0.596/0.595 g versus 0.700/0.706 g in the control group. These effects were considered to be possibly related to the treatment with the test item.

In females at the dose level of 1000 mg/kg bw/day slightly, not statistically significantly higher absolute liver weight was recorded. Liver weight to body weight as well as to brain weight ratios were also increased when compared to the control group; the difference in liver weight to body weight ratio was statistically significant. Changes of liver weights in females were considered to be possibly related to the treatment with the test item.

Remaining changes in organ weights were considered not to be related to the treatment.

In males at the dose level of 1000 mg/kg bw/day, statistically significantly higher kidney weight to body weight ratio was noted in the absence of significant changes of absolute kidney weights or kidney weights to brain weights ratio. This was considered to be a secondary effect to reduced body weights and not an primary effect on kidney weights.

In females at the dose level of 1000 mg/kg bw/day, statistically significantly higher brain weight to body weight ratio was noted whereas absolute brain weights were slightly, not statistically significantly lower when compared to the respective control values. These changes were considered to be a result of lower body weights and not primarily related to the treatment with the test item. Statistically significantly lower absolute heart weights were noted at the dose level of 1000 mg/kg bw/day and statistically significantly lower heart weight to brain weight ratio were noted at the dose levels of 200 and 1000 mg/kg bw/day. Heart weights to body weights ratios were similar at all dose levels and therefore lower heart weights were considered to be most probably related to reduced body weights and not primarily a test item-related effect.

No further statistically significant changes in organ weights were noted at any dose level.

MACROSCOPICAL FINDINGS

No test item-related findings were noted during necropsy of males at any dose level.

All noted findings in males were considered to be within the range of normal background alterations: incompletely collapsed lungs in three males at the high-dose level, foci on lungs in one male in the control group, foci on tongue in one male at the high dose level, foci in stomach of one male at each low-, mid- and high-dose level, pelvic dilation in one male at each low- and mid-dose level, foci on thymus of one male in the control group and four males at mid-dose level and discoloration of mandibular lymph node in one male at the mid-dose level.

No further findings were noted in males at any dose level.

No test item-related findings in females were noted during necropsy of females at any dose level.

All noted findings were considered to be within the range of normal background alterations: incompletely collapsed lungs in one female at the high-dose level, foci in stomach of one female at low-dose level, pelvic dilation in one female at low-dose level, thickened uterus in one female at the low-dose level, missing adrenal gland (unilateral) in one female at the mid-dose level, thymus reduced in size in one female at the mid-dose level and alopecia in one female at the mid-dose level.

No further findings were noted in females at any dose level.

HISTOPATHOLOGY FINDINGS

- Liver: Central to diffuse hepatocellular hypertrophy was recorded at minimal severity in both males and females at the dose level of 1000 mg/kg bw/day.
- Kidneys: Incidence and severity of hyaline droplets in the epithelium were increased in males at the dose level of 1000 mg/kg bw/day.
- Stomach: Squamous hyperplasia of the forestomach was recorded at minimal to slight severity in animals at the dose level of 1000 mg/kg bw/day, and few females at the dose levels of 200 and 40 mg/kg bw/day.
- Thyroid Gland: Follicular cell hypertrophy was recorded at minimal severity in both males and females at the dose level of 1000 mg/kg bw/day.
- Other Findings: No test item-related histological findings were recorded in ovary on females which did not give birth or in the reproductive organs of infertile males.

The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

- Sperm Staging: No differences in the completeness of stages or cell populations of the testes were recorded between controls and high-dose animals.

4. REPRODUCTION AND BREEDING

MATING PERFORMANCE AND FERTILITY

Mating performance and fertility were considered not to be affected by the treatment at any dose level.

Percentage of mating was 100% in all groups. With the exception of one female at the high dose level (no. 79) mating of all females was recorded during the first pairing period. After 14 days of unsuccessful pairing of female no. 79 with male no. 35, a second pairing of this female with male no. 39 was commenced. Mating of this female was confirmed on day two of the pairing with male no. 39.

Mean (median) precoital times calculated for the first pairing period were 2.4 (3), 2.3 (3), 2.8 (3) and 3.8 (4) days in order of ascending dose levels.

One female at the dose level of 40 mg/kg bw/day (no. 59), one female at the dose level of 200 mg/kg bw/day (no. 70) and three females at the dose level of 1000 mg/kg bw/day (nos. 81, 82 and 88) were not pregnant. Consequently, fertility indexes (numbers of females pregnant as percentages of females paired) and conception rates (numbers of females pregnant as percentages of females mated) were 100%, 90.9%, 90.9% and 72.7% at the dose levels of 0, 40, 200 and 1000 mg/kg bw/day, respectively.

Decreased fertility at the high dose level was considered to be possibly related to the treatment with the test item.

CORPORA LUTEA COUNT

No effects on corpora lutea count were observed at any dose level. Mean number of corpora lutea per dam was 11.3, 11.1, 9.8 and 12.1 in order of ascending dose levels.

DURATION OF GESTATION

No effects on duration of gestation were observed at any dose level. Mean duration of gestation was 21.6, 21.9, 22.1 and 22.4 days, in order of ascending dose level.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

At the dose level of 1000 mg/kg bw/day, a lower number of implantations was noted. Mean number of implantations per dam was 8.5 at this dose level, compared to 12.2 in the control group. This difference was not statistically significant but below the historical control range (containing values from 11.4 to 13.7). For this reason reduction in mean number of implantation sites was considered to be related to the treatment with the test item.

Treatment with the test item caused an increase in post-implantation loss at all dose levels with a total post implantation loss in six litters at the high dose level. Mean incidence of post implantation loss per dam was 0.4, 2.0, 3.8 and 8.5 at the dose levels of 0, 40, 200 and 1000 mg/kg bw/day, respectively. The differences to the control value were statistically significant in all dose groups.

LITTER SIZE AT FIRST LITTER CHECK

Treatment with the test item caused reduction of litter size at first litter check in all dose groups.

At the dose level of 1000 mg/kg bw/day, no living pups were found at first litter check.

In two litters (nos. 83 and 87) dead pups were found at first litter check. In remaining litters, beginning of delivery was noticed; first delivered pups were noted or supposed in the cage, but no living pups were found in the cages during the first litter check. It should be considered that at least some of the females at the high dose level delivered their pups but cannibalized them shortly thereafter.

At the dose level of 200 mg/kg bw/day, 24 pups (from 4 litters) were found dead at first litter check whereas mean number of living pups per dam was 8.8. At the dose level of 40 mg/kg bw/day 5 pups (from 4 litters) were found dead at first litter check whereas mean number of living pups per dam was 10.7. In the control group, no dead pups at first litter check were noted; mean number of living pups per dam was 11.9.

Differences at the mid- and low-dose levels were not statistically significant. However, lower litter size resulted from test item-related increase of post-implantation loss and values at these dose levels were beyond the historical control background (containing values of mean number of living pups per dam from 10.3 to 13.2). For these reasons, reduction of litter size at the mid-, and low-dose levels were considered to be test item-related.

In all dose groups, statistically significant reduction in birth index (number of pups born alive as a percentage of implantation sites) was noted. Mean birth index was 96.7%, 84.3%, 68.8% and 0.0% at the dose levels of 0, 40, 200 and 1000 mg/kg bw/day, respectively. This effect was considered to be test item-related.

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

Increased postnatal loss was noted in all dose groups.

At the dose level of 200 mg/kg bw/day, 27 pups (from 5 litters) were lost during lactation.

At the dose level of 40 mg/kg bw/day, 9 pups (from 1 litter) were lost during lactation.

In the control group, one pup was lost during lactation.

Consequently, statistically significant reduction in viability index (number of pups on day 4 post partum as a percentage of pups born alive) was noted in mid- and low-dose groups. Mean viability index was 99.2%, 91.6% and 69.3% at the dose levels of 0, 40 and 200 mg/kg bw/day, respectively. This effect was considered to be test item-related.

Dose descriptor:
NOAEL
Remarks:
for general toxicity
Effect level:
40 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Transient salivation and bedding in mouth as well as slight reduction in food consumption (both genders) and slightly lower body weight gain (females) were considered not to be adverse.
Dose descriptor:
NOAEL
Remarks:
for reproduction/developmenatl toxicity
Basis for effect level:
other: Increased post-implantation loss and postnatal loss as well as reduced litter size noted in all treated groups.
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
No milk in stomach and pup cold to the touch
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
Only dead pups in group 4 at first litter check. Reduced viability index in groups 2 and 3
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
No weights recorded in group 4 - only dead pups. In group 3, reduction in body weights and body weight gain
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION

Following findings were considered to be related to the treatment with the test item: no milk in the stomach and pups cold to touch.

During first litter check, no milk in the stomach was noted in one pup at the dose level of 1000 mg/kg bw/day, 15 pups (dead) (from 2 litters) at the dose level of 200 mg/kg bw/day and 3 pups (from 2 litters) at the dose level of 40 mg/kg bw/day; all these pups were dead at first litter check. At the low-dose level 2 further pups (from 1 litter) had no milk in the stomach on day 2 post partum.

Several pups dead at first litter check: 6 (from 2 litters) at the dose level of 1000 mg/kg bw/day and 4 (from 2 litters) at the dose level of 200 mg/kg bw/day were found partially cannibalized.

In the control group, one fetus was found with malpositioned left hind leg and malpositioned base of tail.

No further findings were noted at first litter check or during the first 4 days post partum in pups at any dose level.

SEX RATIOS

Pups sex ratio was not affected by exposure to the test item at any dose level.

At first litter check, percentages of male pups were 53%, 44% and 51% at the dose levels of 0, 40 and 200 mg/kg bw/day.

BODY WEIGHTS TO DAY 4 POST PARTUM

Because only dead pups were found at the dose level of 1000 mg/kg bw/day, no data on body weights of pups at the high-dose level were obtained.

At the dose level of 200 mg/kg bw/day, lower body weight and lower body weight gain of pups during lactation were noted. The differences to the control value were not statistically significant but reduction in body weights and body weight gain occurred in litters with increased mortality. Because of this correlation effects on body weights and body weight gain in mid-dose group were considered to be test item-related.

Mean body weights of pups on day 1 post partum were: 6.4 g, 6.6 g and 5.9 g and mean body weight gains during lactation were +47.5%, +48.5%, and +40.2%, at the dose levels of 0, 40, and 200 mg/kg/day, respectively.

MACROSCOPICAL FINDINGS

No findings were found during necropsy of pups at any dose level.
Dose descriptor:
LOAEL
Generation:
F1
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
Reproductive effects observed:
not specified

SUMMARY OF PERFORMANCE

 

Table 1: P Animals Breeding for F1 Litters

 

Group
(mg/kg/day)

1
(0)

2
(40)

3
(200)

4
(1000)

Female numbers

45-55

56-66

67-77

78-88

Number of females paired

11

11

11

11

Number of females mated

11

11

11

11

Number of females not pregnant (A)

0

1

1

3

Numbers of females, which lost their litters before first litter check (B)

0

0

1

8

Number of females, which lost their litters during lactation (C)

0

0

1

0

Number of females which reared their pups until day 4 post partum

11

10

8

0

(A)       Female Nos. 59, 70, 81, 82 and 88.

(B)       Female Nos. 68, 78, 79, 80, 83, 84, 85, 86 and 87 had no living pups at first litter check.

(C)       Female No. 73.

Table 2: Percentages of fertility figures

Group
(mg/kg/day)

1
(0)

2
(40)

3
(200)

4
(1000)

 Percentage mating (%)  100.0  100.0 -  100.0 -  100.0 -
 Ferility index (%)  100.0   90.9 -   90.9 - 72.7 - 
 Conception rate (%)  100.0   90.9 -   90.9 -  72.7 -
 Gestation index (%)  100.0  100.0 -   90.0 -   0.0 ##

#/##/- : Fisher's Exact Test, significant at 5% (#), 1% (##) or not significant (-)

+/++/- : Steel Test, significant at 5% (+), 1% (++) or not significant (-)

Table 3: Breeding data

Group
(mg/kg/day)

1
(0)

2
(40)

3
(200)

4
(1000)

Number of litters  11  10  10  8
Duration of gestation        
   Mean  21.6  21.9 -  22.1 -  22.4 -
   ST.Dev  0.5  0.3  0.6  0.7
   N  11  10  10  8
Corpora lutea        
   Total  124  111  88  97
   Mean  11.3  11.1 -  9.8 -  12.1 -
   ST.Dev  2.7  3.9  2.5  5.2
   N  11  10  9  8
 Implantations        
   Total  134  127  109  68
   Mean  12.2  12.7- 12.1-  8.5 -
   St.Dev  4.0  3.2  2.8  3.7
   N  11  10  9  8
 Living pubs at first check        
   Total  131  107  88  0
   Mean  11.9 10.7 -  8.8 -  0 ++
   St.Dev  3.6  2.5  4.0  0.0
   N  11  10  10  8
Living pubs on day 4 post partum        
   Total  130  98  61  0
   Mean  11.8  9.8 -  6.1 +  0.0 ++
   St.Dev  3.5  3.2  4.1 0.0 
   N  11  10  10  8
 Postnatal loss days 0 -4 post partum        
   Total  1  9 ##  27##  0
   Mean  0.1  0.9 -  2.7 -  0.0 -
   St. Dev.  0.3  2.8  3.6  0.0
   N  11  10  10  8
 Post Implantation loss        
   Total  4  20 ##  34 ##  68 ##
   Mean  0.4  2.0 ++  3.8 +  8.5 ++
   St. Dev  0.5  1.6  4.1  3.7
   N  10  10  9  8

#/##/- : Fisher's Exact Test, significant at 5% (#), 1% (##) or not significant (-)

+/++/- : Steel Test, significant at 5% (+), 1% (++) or not significant (-)

Conclusions:
The read-across supporting substance (Pentapropylensuccinimido)-hexanoic acid, sodium and triethanolamine salts induced decrease of body weight and decrease of body weight gain at 200 and 1000 mg/kg bw. At 1000 mg/kg bw reduced fertility index, reduced conception rate, reduced gestation index and increased pre-implantation loss was observed. At 200 mg/kg bw all relevant parameters for the fertility were not affected. No significant effect on the fertility could be derived.
Executive summary:

The reproductive toxicity of the registration substance was assessed based on data on the read-across substances, (Pentapropylensuccinimido)-hexanoic acid, sodium and triethanolamine salts.

 

(Pentapropylensuccinimido)-hexanoic acid, sodium and triethanolamine salts was investigated for its reproductive toxicity according to the OECD Guideline 422. The applies doses were 0, 40, 200 and 1000 mg/kg bw, using water as vehicle.

No mortality and no apparent signs for the clinical observation was found as well as no effect in the functional observational battery. Effects on food consumption and body weight development were observed dose dependently. The effects at 1000 and 200 mg/kg bw were considered to be indicative of adverse effects.

At dose of 1000 mg/kg bw, changes in clinical chemistry and hematology parameters comprised increased prothormbin time, increased potassium level and increased total protein. No comparable effects were found for 200 or 40 mg/kg bw.

At dose of 1000 mg/kg bw, increased liver and spleen weights were found. No comparable effects were found for 200 or 40 mg/kg bw.

There was no macroscopic finding for all treated animals. The histopathological alteration comprised central to diffuse hepatocellular hypertrophy in males and females of 1000 mg/kg bw, increased incidence and severity of hyaline droplets in the kidney epithelium in males of 1000 mg/kg bw, squamous hyperplasia of the forestomach that occurred down to the 40 mg/kg bw group and follicular cell hypertrophy in thyroid at 1000 mg/kg bw.

The NOALE of 40 mg/kg bw was obtained.

As for the fertility of the parental animals no effect was found on the reproduction organs in all treated animals.

At 1000 mg/kg bw, fertility index, conception rate and gestation index were reduced and the pre-implantation loss increased. At 200 mg/kg bw all relevant parameters for the assessment of the fertility (effects up to the implantation) were not affected.

As for the developmental toxicity, increased post-implantation loss and increased post natal loss were observed down to the lowest dose level of 40 mg/kg bw. The LOAEL of 40 mg/kg bw is assigned for the developmental toxicity.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
40 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One valid study available
Additional information

Justification for read-across approach:

The registraton substance is a weak acid and the read-across supporting substance is the corresponding weak base. When dissolved in water the read-across supporting substance will be converted to the registration substance.

Study summary

The read-across supporting substance (Pentapropylensuccinimido)-hexanoic acid, sodium and triethanolamine salts was investigated for its reproductive toxicity according to the OECD Guideline 422. The applied doses were 0, 40, 200 and 1000 mg/kg bw.

No mortality and no apparent signs for the clinical observation was found as well as no effect in the functional observational battery. Effects on food consumption and body weight development were observed dose dependently. The effects at 1000 and 200 mg/kg bw were evaluated as indications of toxicity.

At 1000 mg/kg bw, changes in clinical chemistry and hematology parameters comprised increased prothormbin time, increased potassium level and increased total protein. No comparable effects were found for 200 or 40 mg/kg bw.

At 1000 mg/kg bw, increased liver weight and increased spleen weight were found. No comparable effects were found for 200 or 40 mg/kg bw.

There was no macroscopic finding for all treated animals. The histopathological alteration comprised central to diffuse hepatocellular hypertrophy in males and females of 1000 mg/kg bw, increased incidence and severity of hyaline droplets in the kidney epithelium in males of 1000 mg/kg bw and follicular cell hypertrophy in thyroid at 1000 mg/kg bw.Further,squamous hyperplasia of the forestomach was found down to the 40 mg/kg bw group.

The NOALE of 40 mg/kg bw was obtained for systemic toxicity.

As for the fertility of the parental animals no effect was found on the reproduction organs in all treated animals.

At 1000 mg/kg bw, fertility index, conception rate and gestation index were reduced and the pre-implantation loss increased. At 200 mg/kg bw all relevant parameters for the assessment of the fertility (effects up to the implantation) were not affected.

As for the developmental toxicity, increased post-implantation loss and increased post natal loss were observed down to the lowest dose level of 40 mg/kg bw. The LOAEL of 40 mg/kg bw is assigned for the developmental toxicity.


Short description of key information:
The reproduction toxicity of the registration substance is assessed based on the data for the read-across supporting substance.
(Pentapropylensuccinimido)-hexanoate, sodium and triethanolamine salt was investigated for its reproduction toxicity potential according to the OECD Guidline 422. Down to dose level of 40 mg/kg bw clear fetoxicities were found. The LOAEL of 40 mg/kg bw was obtained.

Justification for selection of Effect on fertility via oral route:
Guideline study; well-performed and well-documented

Effects on developmental toxicity

Description of key information
The read-across supporting substance (Pentapropylensuccinimido)-hexanoic acid, sodium and triethanolamine salts was investigated equivalent to the OECD Guideline 414.  Clear fetotoxicities were observed at doses that were associated with apparent maternal toxicity.   The LOAEL of 8 mg/kg bw was obtained.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2012-2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Scientifically well-performed; obtained result sufficiently reliable and robust for classification of the registration substance; equivalent to OECD 414 with reduced number of animals; Non-GLP Justification for read-across: the grouped substances differ only that the registraton substance is a weak acid and the read-across substance is the corresponding weak base. When dissolved in water the read-across supporting substance will be converted to the registration substance.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
reduced number of animals; 5 animals per group instead of 20
Principles of method if other than guideline:
Aim of the study: In the OECD 422 study on the registration substance, reduced number of pubs were found. The introduced study aimed to clarify whether the effects found in the OECD 422 originated from the fertility impairment or fetotoxicity. The use of reduced number of animals is justified, since the study did not aim to demonstrate the absence of the reproduction toxicity.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Wistar
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
no
Details on mating procedure:
After acclimatization, females will be housed with sexually mature males (1:1) in special automatic mating cages i.e. with synchronized timing to initiate the nightly mating period, until evidence of copulation is observed. This system reduces the variation in the copulation times of the different females. The females will be removed and housed individually if:
- the daily vaginal smear is sperm positive, or
- a copulation plug is observed.
The day of mating will be designated day 0 post coitum.
Male rats of the same source and strain will be used only for mating. These male rats are not considered part of the test system. The fertility of these males has been proven and is continuously monitored.
Duration of treatment / exposure:
day 6 post coitum onwards; last treatment on day 20 post coitum
Frequency of treatment:
once per day
Duration of test:
Caesarean section and necropsy on day 21 post coitum
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Maternal examinations:
Viability / Mortality: twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimation and up to day of necropsy)
Food Consumption: recorded at 3-day intervals
Body Weights: recorded daily from day 0 until day 21 post coitum
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
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [half per litter ]
- Skeletal examinations: Yes: [half per litter ]
- Head examinations: Yes: [half per litter ]
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes
Key result
Dose descriptor:
LOAEL
Effect level:
8 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
Key result
Abnormalities:
effects observed, treatment-related
Description (incidence and severity):
Changes in the size or shape of the nasopharyns, nasal septum incompletely fused to the palate at dose of 200 mg/kg bw; Increased number of supernumerary ribs at 40 and 200 mg/kg bw: Small spleen down to dose of 8 mg/kg bw.
Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
8 mg/kg bw/day
Treatment related:
yes
Relation to maternal toxicity:
developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Study Summary:

Study design: The study was performed equivalent to the OECD Guideline 414 with the deviation that 5 pregnant animals per dose group were used. The application route was per gavage with water as vehicle. The applied doses were 0, 8, 40 and 200 mg/kg bw. The observations parameters were according to the Guideline as well.

Maternal toxicity: Upon clinical observation no effect indicative of toxicity was found. At 200 mg/kg bw the food consumption and the body weight gain were slightly reduced. No effect was found for animals of 40 and 8 mg/kg bw.

Reproduction data upon Caesarian section: No difference was found for control and treated animals upon investigation on number of corpora lutea, implantation sites and number of live fetuses.

Fetal evaluation:

-         External examination: two fetuses of 200 mg/kg bw exhibited abnormalties; one no lower jaw, small mouth opening and possibly a cleft palate and the other a cleft palate.

-         Visceral abnormalities: all fetuses of 200 mg/kg bw had small spleen and incomplete fusion of nasal septum to palate; small spleen was found down to dose level of 8 mg/kg bw.

-         Skeletal abnormalities: abnormalities were found in all fetuses of 200 mg/kg bw, comprising thin skull zygomatic jugal arch, abnormal curvature of pectoral girdle clavicle, absent humerus deltoid tuberosity in forelimb, short mid region of rib cage, abnormal curvature of hyoid body and abnormal spacing of zygomatic arch structures; increased number of supernumerary ribs was found at 200 and 40 mg/kg bw.

Tab: Incidence of some fetal toxicity found down to mid/low doses

a) Small spleen in intrauterine exposed fetuses

 

Control

n=32

8 mg/kg bw

n=36

40 mg/kg bw

n=35

200 mg/kg bw

n=32

Spleen small or small severe (total)

1

(3%)

4

(11%)

17

(49%)

32

(100%)

     Small (ca. 75% of expected size)

1

(3%)

4

(11%)

16

(46%)

5

(16%)

     Small severe (ca. 50% of expected size)

0

(-)

0

(-)

1

(3%)

27

(84%)

b) Supernumerary ribs, rudimentary in intrauterine exposed fetuses

 

Control

n=29

8 mg/kg bw

n=33

40 mg/kg bw

n=33

200 mg/kg bw

n=28

Left

7

(24%)

8

(24%)

26

(79%)

24

(82%)

Right

8

(28%)

5

(15%)

24

(73%)

25

(89%)

 

Conclusion: Clear toxic effects were found for fetuses at doses that were not associated with an apparent maternal toxicity.

Conclusions:
The registration substance was assessed based on the data on read-across supporting substance, in which clear fetotoxicity was found at doses that were not associated with apparent maternal toxicities. The study is considered to be valid to demontrate the developmental toxicity of the registration substance. The assignment of Cat 2:R61 is justified according to 67/548/EEC (DSD).
Executive summary:

The developmental toxicity of the registration substance was assessed based on data on the read-across substances, (Pentapropylensuccinimido)-hexanoic acid, sodium and triethanolamine salts.

(Pentapropylensuccinimido)-hexanoic acid, sodium and triethanolamine salts was investigated for its developmental toxicity equivalent to the OECD Guideline 414 with the deviation that 5 pregnant animals per dose group were used. The application route was per gavage with water as vehicle. The applied doses were 0, 8, 40 and 200 mg/kg bw. The observations parameters were according to the Guideline as well.

No significant clinical effect was found for all treated dams. At 200 mg/kg bw the food consumption and the body weight gain were slightly reduced. No effect was found for animals of 40 and 8 mg/kg bw.

With respective to the reproduction performance of the treated animals, the investigated parameters such as number of corpora lutea, implantation sites and number of live fetuses were comparable to those of control animals.

Upon examination of fetuses, increased frequency of a small spleen was noted down to the dose level of 8 mg/kg bw/day. Increased number of supernumerary ribs (rudimentary) was noted at the dose levels of 200 and 40 mg/kg bw/day. In addition, changes in the size or shape of the nasopharyns, nasal septum incompletely fused to the palate as well as several abnormalitiers and variations in skeleton were noted at the dose level of 200 mg/kg bw/day.

Based on the fetotoxicity found down to dose of 8 mg/kg bw, the LOAEL of 8 mg/kg bw was established.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
8 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Two studies on read-across supporting substance are available. The obtained results are consistent
Additional information

Justification for read-across appraoch:

The registraton substance is a weak acid and the read-across supporting substance is the corresponding weak base. When dissolved in water the read-across supporting substance will be converted to the registration substance.

Study summary

The read-across supporting substance (Pentapropylensuccinimido)-hexanoic acid, sodium and triethanolamine salts was investigated for its developmental toxicity equivalent to the OECD Guideline 414 with the deviation that 5 pregnant animals per dose group were used. The application route was per gavage with water as vehicle. The applied doses were 0, 8, 40 and 200 mg/kg bw. The observations parameters were according to the Guideline as well.

No clinical effect was found for all treated animals. At 200 mg/kg bw the food consumption and the body weight gain were slightly reduced. No effect was found for animals of 40 and 8 mg/kg bw.

With respective to the reproduction performance of the treated animals, the investigated parameters such as number of corpora lutea, implantation sites and number of live fetuses were comparable to those of control animals.

During examination of fetuses, increased frequency of a small spleen was noted down to the dose level of 8 mg/kg bw/day. Increased number of supernumerary ribs (rudimentary) was noted at the dose levels of 200 and 40 mg/kg bw/day. In addition, changes in the size or shape of the nasopharyns, nasal septum incompletely fused to the palate as well as several abnormalitiers and variations in skeleton were noted at the dose level of 200 mg/kg bw/day.

Based on the fetotoxicity found down to dose of 8 mg/kg bw, the LOAEL of 8 mg/kg bw was established.

Use of the obtained NOAEL/LOAEL for the DNEL derivation.

The registraton substance is a weak acid and the read-across supporting substance is the corresponding weak base. When dissolved in water the read-across supporting substance will be converted to the registration substance

Since the conversion from salt to acid form is likely to occur already in the gastro-intestinal tract when given orally to the animals, there should be no bioavailability difference based on the molar dose. For the NOAEL for the acid form the only factor to be considered is the molecular mass difference (420 g/mol vs ca. 530 g/mol). In the presented key study, the applied doses were 8, 40, 200 mg/kg bw, corresponding to 6, 30 and 160 mg/kg bw for the acid form. For the DNEL derivation the dose levels of 6 mg/kg bw as LOAEL will be used.

 


Justification for selection of Effect on developmental toxicity: via oral route:
Scientifically well-performed; obtained result sufficiently reliable and robust for classification of the registration substance; equivalent to OECD 414 with reduced number of animals; Non-GLP

Justification for classification or non-classification

The registration substance should be classified according to DSD 67/548/EEC as

T, Reprotox, Cat2; R1

Rationale for assigning Cat 2: R61 for the endpoint developmental toxicity

In the available developmental toxicity study equivalent to OECD 414, clear fetotoxicities were observed at doses that were not associated with apparent maternal toxicity. The assignment of Cat 2:R61 is justified according to 67/548/EEC (DSD).

 

Rationale for  “no classification” for the endpoint fertility

In the available OECD 422 study, no significant effect was found on the reproduction organs in all treated animals.

At 1000 mg/kg bw, fertility index, conception rate and gestation index were reduced and the pre-implantation loss increased. At 200 mg/kg bw all relevant parameters for the assessment of the fertility (effects up to the implantation) were not affected.

Since the impairment of the body weight development in the pre-mating phase was apparent at 200 and 1000 mg/kg bw, “no classification” is justified.