Fatty acids, C18-unsatd., esters with triethanolamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction

Remarks:

other: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-Dec-2012 to 06-Sep-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source:Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 Weeks
- Weight at study initiation: Males: 299 to 335g, Females: 209 to 252g
- Housing: Animals were housed Individually in Makrolon type-3 cages with wire mesh tops and standard softwood bedding including paper enrichment
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2018C
- Water (e.g. ad libitum): Community tap-water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C;
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

DOSE FORMULATIONS

The dose formulations were prepared weekly.

The test item was weighed into a glass beaker on a tared Mettler balance. The vehicle was added and the mixtures were stirred using a magnetic stirrer and used at room temperature (20 ± 5 °C). Separate formulations were prepared for each concentration. Each formulation was divided in daily aliquots.

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

STABILITY AND STORAGE OF DOSE FORMULATION

- Stability of Dose Formulations: 8 days
- Storage of Dose Formulations: At room temperature (20 ± 5 °C) in glass beakers

TREATMENT
- Frequency of Administration: Daily, at approximately 24 hour intervals
- Dose Levels: 0 mg/kg/day (Group 1), 100 mg/kg/day (Group 2), 300 mg/kg/day (Group 3) and 1000 mg/kg/day (Group 4)
- Rationale for Dose Level Selection: Dose levels were selected based on the results of a dose range-finding study
- Dose Volume: 5 mL/kg body weight
- Dose Calculation: The daily doses were calculated and adjusted according to the daily determined body weights of the individual animals.
- Dose Concentrations: 0 mg/mL (Group 1), 20 mg/mL (Group 2), 60 mg/mL (Group 3) and 200 mg/mL (Group 4)
- Duration of Treatment Period: 46 days (males) and 40 to 56 days (females)

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) of the same treatment group 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 done.

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:

The dose formulations were analyzed using a HPLC method.

Concentration, homogeneity and stability of dose formulations were determined in samples taken after experimental start and in samples taken during week 6 of treatment

The test item was used as analytical standard.

RESULTS

The test item concentrations in the dose formulations ranged from 89.1% to 106.1% with reference to the nominal and were within the accepted range of ±20%.

The homogeneous distribution of test item in the preparations was approved because single results found did not deviate more than 5.4% from the corresponding mean and met the specified acceptance criterion of ≤15%.

In addition, the test item was found to be stable in application formulations when kept 8 days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value, except for group 2 that slightly exceeded the acceptance criteria (up to 1.2%). However, as no degradation was observed, the formulation is considered to be stable, although not fulfilled the acceptance criterion (±10%).

In conclusion, the results indicate the accurate preparation and storage of the test item in vehicle during this study.

Duration of treatment / exposure:

Administered to male rats for 46 days and to female rats for a period of 40 to 56 days.

Frequency of treatment:

Once daily

Details on study schedule:

SCHEDULE FOR MALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 19 days maximum
- Treatment End: On day before sacrifice
- Necropsy: After treatment when no longer needed for assessment of reproductive effects

SCHEDULE FOR FEMALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 19 days maximum
- Gestation: Approximately 21 days
- Treatment End: On day 4 post partum
- Necropsy: On day 4 post partum (pups on day 4 post partum)

Remarks:

Doses / Concentrations:
0 (control), 100 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 males and 12 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected in agreement with the Sponsor, based on the results of a dose range-finding study.

Positive control:

Not required

Parental animals: Observations and examinations:

VIABILITY / MORTALITY

Twice daily

CLINICAL SIGNS

Daily cage-side clinical observations were performed once daily during acclimatization period. Thereafter, up to the necropsy, daily clinical observation was performed immediately before dosing, up to 30 minutes post-dosing and one hour after dosing. An additional observation was made five hours after dosing during the normal working week. Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION

- Males: Weekly during pre-pairing and after pairing periods
- Females: Pre-pairing period days 1 - 8 and 8 - 13; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum.

No food consumption was recorded during the pairing period.

WATER CONSUMPTION

Monitored daily by visual inspection of water bottles.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy

DETAILED CLINICAL OBSERVATIONS

Detailed clinical observations were performed outside the home cage in all P generation animals. In all males of P generation it was performed once prior to the first administration of the test item and weekly thereafter. In females of P generation, it was prepared once prior to the first administration of the test item, 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 post partum) relevant parameters were performed with 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, constitution, skin, pupil 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, rectal temperature.

Additionally, locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded.

Low beams count was reported at 10-minute intervals as well as the total activity over the measuring period.

CLINICAL LABORATORY INVESTIGATIONS

Blood samples were obtained at the end of the pre-pairing period from 5 males and 5 females from each group. 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.


Hematology:
The following hematology parameters were determined:

Complete Blood Cell Count:

- 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
- Reticulocytes

Coagulation:

- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

Clinical Biochemistry:
The following clinical biochemistry parameters were determined:

- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio
- Bile acids

Oestrous cyclicity (parental animals):

Not examined

Sperm parameters (parental animals):

Not examined

Litter observations:

The litters were examined daily 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) and (with identification) on days 1 and 4 post partum. In addition, a surface righting assessment was performed on day 1 post partum.

Postmortem examinations (parental animals):

TERMINATION AND NECROPSY

Dams were sacrificed on day 4 post partum. Males were sacrificed when they were no longer needed for the assessment of reproductive effects.

If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

For the parent animals, special attention was directed at the organs of the reproductive system. 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, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken:

From all parental males:
- Testes (as pairs)
- Epididymides (as pairs)
- Seminal vesicles (as pairs)
- Prostate

From all parental females:
- Ovaries (as pairs)
- Uterus and Cervix

From all parental animals of both genders:
- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Pituitary
- Thymus
- Spleen
- Thyroid

TISSUE PRESERVATION

Tissues from the following reproductive organs from all parental males were preserved in neutral phosphate buffered 4% formaldehyde solution (or in Bouin’s fixative, if indicated):
Prostate
- Seminal vesicles with coagulating gland
- Testes (in Bouin’s fixative)
- Epididymides (in Bouin’s fixative)

Tissues from the following reproductive organs from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries
- Uterus
- Vagina
- Cervix

In addition, from all males and females at scheduled necropsy 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 (including cerebrum, cerebellum and pons)
- Spinal cord (cervical, mid-thoracic and lumbar)
- Small and large intestines (caecum, colon, duodenum, rectum, ileum and jejunum incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Aorta (thoracic)
- Bone and bone marrow (femur including stifle joint)
- Bone and bone marrow (sternum)
- Muscle (skeletal)
- Oesophagus
- Parathyroid
- Pancreas
- Pituitary
- Salivary glands (sublingual / mandibular)
- Skin (abdominal region)
- Eyes (fixed in Davidson's fluid)
- Mammary tissue

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 collected at terminal sacrifice from the first five males and first five females which gave birth and slides of all reproductive organs from all males and females 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 and to all unfertile animals.

Qualitative assessment of the male reproductive organs was performed. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. Histological examination of ovaries was carried out on any females that did not give birth. In addition, microscopic examination of the reproductive organs of male 35 and of infertile males was made (except for males no. 18 and 34 from which no reproductive organs were processed and examined by error).

A histopathology peer review was performed.

Postmortem examinations (offspring):

Pups were sacrificed by an injection of sodium pentobarbital on day 4 post partum. Dead pups, except those excessively cannibalized, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, food consumption, body weight, macroscopical findings, organ weights and ratios, clinical laboratory data, as well as reproduction data:

- 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.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated: mean precoital time, percentage mating, fertility index, conception rate, pre and post-implantation loss, gestation index, birth index and viability index.

Offspring viability indices:

From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex ratio and viability index.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Mucosal parakeratosis of the vagina was observed in 3 out of 4 non pregnant females treated at 1000 mg/kg bw/day. A relationship with the test item treatment cannot be excluded.

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

1. IN-LIFE DATA

VIABILITY / MORTALITY

No test item-related premature deaths were recorded.

Male no. 4 (0 mg/kg bw/day, control group) was sacrificed on day 19 of the pairing period due to severe clinical signs which included muscular hypotonus (grade 3 of 3), decreased activity (grade 3 of 3), hunched posture, ruffled fur (grade 2 of 3), and reddish nasal secretion (grade 3 of 3). At necropsy, several reddish spots in the stomach mucosa were noted. Histopathologically no abnormalities were noted which could explain the cause of the moribund condition of the animal.

Female no. 61 (100 mg/kg bw/day) was found dead on day 1 of the gestation period. An abnormal gait, sedation (grade 2 of 3), hunched posture, ruffled fur (grade 2 of 3), and tachypnea (grade 2 of 3) were observed before its premature death. The main microscopic finding was a marked diffuse acute inflammation in the lungs that was indicative of a gavage accident.

GENERAL CAGESIDE OBSERVATIONS (DAILY)

No test item-related clinical signs were found at any dose level.

A kinked tail apex, hair loss, vaginal bleeding and salivation were observed in individual animals and were considered to be incidental findings and not treatment-related.

DETAILED CLINICAL OBSERVATIONS (WEEKLY)

No test item-related clinical signs were found at any dose level.

A kinked tail apex was considered to be an incidental finding and not treatment-related.

FUNCTIONAL OBSERVATIONAL BATTERY

No test item-related effects on behavior, reflexes, grip strength, body temperature and locomotor activity were observed.

Statistically significant decreased body temperatures in all test item-treated female groups were considered to be caused by unusually high body temperatures in the control groups. Furthermore, the decrease was not dose-related and the values recorded in the test item-treated groups were within the range of the historical controls.

Statistically significant increased locomotor activity in males treated with 1000 mg/kg bw/day was considered to be incidental, because the effect was restricted to the first two 10 min intervals, not dose-related and no other findings (e.g. behavioral abnormalities) correlated with this effect.

Statistically significant increased locomotor activity in females treated with 100 mg/kg bw/day was considered to be incidental, because the effect was not observed at higher dose levels.

FOOD CONSUMPTION OF MALES

No effects on food consumption of males were observed at any dose level.

FOOD CONSUMPTION OF FEMALES

No effects on food consumption of females were observed at any dose level.

BODY WEIGHTS OF MALES

No effects on body weight development in males were recorded.

BODY WEIGHTS OF FEMALES

No effects on body weight development in females were recorded.

2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

No effects on hematology parameters were recorded.

A change in a single parameter achieving individual statistical significance was considered to be unrelated to treatment, because it lacked a dose-relationship and was clearly within the historical control range.

CLINICAL BIOCHEMISTRY

No effects on biochemistry parameters were recorded.

Changes in several parameters achieving individual statistical significance were considered to be unrelated to treatment, because they lacked a dose-relationship and/or were clearly within the historical control range.

3. TERMINAL FINDINGS

ORGAN WEIGHTS

No test-item related effects on organ weights were observed.

MACROSCOPIC FINDINGS

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

Reduced size of testes/epididymides in 1/12 males (no.37) recorded at 1000 mg/kg bw/day is a common incidental finding in rats of this age and strain.

All other findings were those commonly seen in the laboratory rat.

MICROSCOPIC FINDINGS

- Males: There were no test item-related findings in any examined organs including reproductive organs. In particular, the qualitative examination of the stages of spermatogenesis in the testis did not reveal any test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. The reduced size of testes/epididymides observed in rat no.37 (1000 mg/kg bw/day) was correlated histologically with moderate multifocal testicular germ cell degeneration leading to moderate abnormal germinal cell content in the epididymides. In the absence of any test-item related effects on male reproductive organs (organ weights or other microscopic change) this isolated finding, known to occur spontaneously in rat, was considered to be fortuitous.

- Non Pregnant Females: Mucosal parakeratosis of the vagina was observed in 3 out of 4 non pregnant females treated with 1000 mg/kg bw/day. The change was characterized by a moderately thickened vacuolated epithelium showing early keratinization and desquamation of parakeratotic cells. In addition, the change was diffuse, more or less homogeneous, and involved areas away from the vaginal opening up to the cervix. The uterine endometrium was lined by tall columnar finely vacuolated cells while the ovary was unremarkable. This finding was considered to be of uncertain toxicological significance. In the remaining non pregnant female of the 1000 mg/kg group, neutrophilic exudative inflammation was present in the uterus and was associated with suppurative exudate in the cervix and vagina; this was consistent with ascending bacterial infection, unrelated to the test item.
There were no test item-related findings in any of the 3 or 1 non pregnant females treated with 100 and 300 mg/kg bw/day respectively.

- Pregnant Females: There were no test item-related microscopic findings, including following evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

4. REPRODUCTION AND BREEDING

MATING PERFORMANCE AND FERTILITY

No test item-related effect on mating performance or fertility was observed at any dose level.

10, 10, 10, and 12 females mated in groups 1, 2, 3, and 4, respectively, within twelve days after initiation of pairing. Mean (median) precoital times were 2.3 (1), 2.5 (3), 4.2 (4) and 2.5 (3) days at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively. All six remaining females mated within 4 days of the second pairing period.

Eight females were not pregnant; three at the low dose level (female nos. 66, 68 and 70 mated with male nos. 13, 19, 20 and 22, respectively), one at the mid dose level (female no. 76 mated with male no. 27) and four at the high dose level (female nos. 85, 87, 90, and 91 mated with male nos. 37, 39, 42, and 43, respectively).

Consequently, fertility indices (number of females achieving pregnancy as a percentage of females paired) were 100%, 73%, 92% and 67% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

The incidence of infertility did not follow a dose dependency and the number of non-pregnant females per group was in the range of the historical background, up to four non-pregnant females were noted in historical control groups. For these reasons lack of pregnancy was considered not to be related to the treatment with the test item.

Except for one female at 300 mg/kg bw/day all pregnant females gave birth to living pups. Consequently, the gestation index (number of females with living pups as a percentage of females pregnant) was 100%, 100%, 91%, and 100% in groups 1, 2, 3, and 4, respectively.

PRE-IMPLANTATION LOSS

No test item-related effects on pre-implantation loss were observed at any dose level.

Mean pre-implantation loss per dam was 1.3, 0.9, 1.1 and 2.0 at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

CORPORA LUTEA COUNT

No effects on corpora lutea count were observed at any dose level.

Mean number of corpora lutea per dam was 14.7, 15.3, 15.5 and 14.4 at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

DURATION OF GESTATION

No effects on duration of gestation were observed at any dose level.

Mean duration of gestation was 21.9, 21.5, 21.6 and 22.0 days at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

IMPLANTATION RATE AND POST IMPLANTATION LOSS

No effects of the test item on implantation rate or post implantation loss were observed at any dose level.

Mean number of implantations per dam was 13.3, 14.4, 14.4 and 12.4 whereas mean number of post-implantation loss per dam was 1.5, 3.4, 1.4 and 2.5; both cited in order of ascending dose levels.

LITTER SIZE AT FIRST LITTER CHECK

No effects on litter size were observed at any dose level.

Mean numbers of living pups per dam at first litter check were 11.8, 11.0, 13.2 and 9.9, whereas birth indexes (number of pups borne alive as a percentage of implantations) were 88.8%, 76.5%, 91.7% and 79.8% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

Dose descriptor:

NOEL

Remarks:

reproduction/developmental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproduction and development were not affected by the treatment.

Remarks on result:

other: Generation: P/F1 (migrated information)

Dose descriptor:

NOEL

Remarks:

general toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Dose descriptor:

NOEL

Remarks:

general toxicity

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

POSTNATAL LOSS DAYS 0 – 4 POST PARTUM

No effects on postnatal loss were observed at any dose level.

In the control group, one pup (from litter no. 52) was missing on day 3 post partum.

At the dose level of 1000 mg/kg bw/day, one pup (from litter no. 86) was found dead on day 1 post partum and one pup (from litter no. 94) was missing on day 1 post partum.

No further mortality of pups was observed at any dose level. Because the incidence of pups mortality did not correlate with the dose levels, the postnatal loss was considered not to be test item-related.

EXTERNAL EXAMINATION OF PUPS AT FIRST LITTER CHECK AND DURING LACTATION

No test item-related findings were noted in pups at any dose level.

PUP 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%, 45%, 47% and 53%, in order of ascending dose level.

RIGHTING REFLEX OF PUPS

No effects on righting reflex of pups were observed at any dose level.

Percentages of pups for which righting reflex was observed within the first one minute of the test were 86%, 84%, 83% and 96% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

BODY WEIGHTS OF PUPS TO DAY 4 POST PARTUM

No effects on pups body weights or body weight gain were noted at any dose level.

MACROSCOPIC FINDINGS OF PUPS

No findings were noted during the necropsy of pups at any dose level.

Reproductive effects observed:

not specified

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litters

 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	49 - 60	61 - 72	73 - 84	85 - 96
Number of females paired	12	12	12	12
Number of females mated	12	12	12	12
Number of females, which died before the scheduled necropsy (A)	0	1	0	0
Numbers of females which did not deliver any pups (B)	0	3	2	4
Number of females which reared their pups until day 4post partum	12	8	10	7

 

(A) Female No. 61 died spontaneously on day 1post coitum

(B) Female Nos. 66, 68, 70, 76, 82 (had 2 implantation sites), 85, 87, 90, and 91

Conclusions:

The NOEL (No Observed Effect Level) for general toxicity in males and for reproduction/developmental toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest dose level used. Due to finding of mucosal parakeratosis of the vagina in females at 1000 mg/kg bw/day, the NOEL for general toxicity in females was considered to be 300 mg/kg bw/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test item 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.

 

The test was administered to male rats for 46 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 for a period of 40 to 56 days.

 

The following dose levels were applied:

 

Group 1:      0 mg/kg body weight/day (control group)

Group 2:  100 mg/kg body weight/day

Group 3:  300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (Arachis oil).

 

The following results were obtained:

PARENT ANIMALS:

 

MORTALITY AND GENERAL TOLERABILITY

 

No test item-related premature deaths were recorded.

 

No test item-related findings were noted during daily clinical or detailed weekly clinical observations in males or females at any dose level.

 

FUNCTIONAL OBSERVATIONAL BATTERY

 

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

 

FOOD CONSUMPTION

 

No effects on food consumption of males or females were observed at any dose level.

 

BODY WEIGHTS

 

No effects on body weights or body weight gain of males or females were observed at any dose level.

 

CLINICAL LABORATORY INVESTIGATIONS

 

No test item-related effects on hematology or clinical biochemistry parameters were noted in males or females at any dose level.

 

REPRODUCTION AND BREEDING DATA

 

No test item-related effect on mating performance, fertility, duration of gestation, corpora lutea count, pre-implantation loss, implantation rate, post implantation and postnatal loss or litter size were noted at any dose level.

 

ORGAN WEIGHTS

 

No test item-related effects on absolute organ weights or organ weights relative to body or brain weights were noted in males or females at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS

 

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

 

There were no test item-related findings in any examined organs including reproductive organs in all males and all pregnant females.

Mucosal parakeratosis of the vagina was observed in 3 out of 4 non pregnant females treated with 1000 mg/kg bw/day. This change was diffuse, more or less homogeneous, and involved areas away from the vaginal opening up to the cervix. Although no other findings correlated with the parakeratosis, a relationship with the test item treatment cannot be excluded. There were no test item-related findings in any of the 3 non pregnant females treated with 100 mg/kg bw/day or the 1 non pregnant female treated with 300 mg/kg bw/day.

 

LITTER DATA - F1 PUPS

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

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

 

No effects on righting reflex were observed in pups at any dose level.

 

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

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 

No effects on pups body weights or body weight gain were noted at any dose level.

 

MACROSCPICAL FINDINGS IN PUPS

 

No findings were noted during the necropsy of pups at any dose level.

 

 

CONCLUSION

 

Based on these results, the NOEL (No Observed Effect Level) for general toxicity in males and for reproduction/developmental toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest dose level used. Due to finding of mucosal parakeratosis of the vagina in females at 1000 mg/kg bw/day, the NOEL for general toxicity in females was considered to be 300 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study has been conducted according to OECD Guideline 422 and GLP and is adequately reported. The study has been assigned a reliability of 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Han Wistar Rat

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats. The test item was administered in Arachis oil as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test item was administered to male rats for 46 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.

Reproduction was not affected by the treatment. Mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, pre and post implantation and postnatal loss or litter size were similar in the control and all dose groups.

The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level used.

Short description of key information:
Reproduction was not affected by the treatment. The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
The available OECD 422 study is assigned as reliability study 1 and is the only reproductive toxicity study available.

Effects on developmental toxicity

Description of key information

Development was not affected by the treatment. The NOEL (No Observed Effect Level)  for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day,

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-Dec-2012 to 06-Sep-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

- Source:Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: 11 Weeks
- Weight at study initiation: Males: 299 to 335g, Females: 209 to 252g
- Housing: Animals were housed Individually in Makrolon type-3 cages with wire mesh tops and standard softwood bedding including paper enrichment
- Diet (e.g. ad libitum): Pelleted standard Harlan Teklad 2018C
- Water (e.g. ad libitum): Community tap-water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C;
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

DOSE FORMULATIONS

The dose formulations were prepared weekly.

The test item was weighed into a glass beaker on a tared Mettler balance. The vehicle was added and the mixtures were stirred using a magnetic stirrer and used at room temperature (20 ± 5 °C). Separate formulations were prepared for each concentration. Each formulation was divided in daily aliquots.

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

STABILITY AND STORAGE OF DOSE FORMULATION

- Stability of Dose Formulations: 8 days
- Storage of Dose Formulations: At room temperature (20 ± 5 °C) in glass beakers

TREATMENT
- Frequency of Administration: Daily, at approximately 24 hour intervals
- Dose Levels: 0 mg/kg/day (Group 1), 100 mg/kg/day (Group 2), 300 mg/kg/day (Group 3) and 1000 mg/kg/day (Group 4)
- Rationale for Dose Level Selection: Dose levels were selected based on the results of a dose range-finding study
- Dose Volume: 5 mL/kg body weight
- Dose Calculation: The daily doses were calculated and adjusted according to the daily determined body weights of the individual animals.
- Dose Concentrations: 0 mg/mL (Group 1), 20 mg/mL (Group 2), 60 mg/mL (Group 3) and 200 mg/mL (Group 4)
- Duration of Treatment Period: 46 days (males) and 40 to 56 days (females)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations were analyzed using a HPLC method.

Concentration, homogeneity and stability of dose formulations were determined in samples taken after experimental start and in samples taken during week 6 of treatment

The test item was used as analytical standard.

RESULTS

The test item concentrations in the dose formulations ranged from 89.1% to 106.1% with reference to the nominal and were within the accepted range of ±20%.

The homogeneous distribution of test item in the preparations was approved because single results found did not deviate more than 5.4% from the corresponding mean and met the specified acceptance criterion of ≤15%.

In addition, the test item was found to be stable in application formulations when kept 8 days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value, except for group 2 that slightly exceeded the acceptance criteria (up to 1.2%). However, as no degradation was observed, the formulation is considered to be stable, although not fulfilled the acceptance criterion (±10%).

In conclusion, the results indicate the accurate preparation and storage of the test item in vehicle during this study.

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) of the same treatment group 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 done.

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.

Duration of treatment / exposure:

Administered to male rats for 46 days and to female rats for a period of 40 to 56 days.

Frequency of treatment:

Once daily

Duration of test:

SCHEDULE FOR MALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 19 days maximum
- Treatment End: On day before sacrifice
- Necropsy: After treatment when no longer needed for assessment of reproductive effects

SCHEDULE FOR FEMALES

- Acclimatization: 7 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 19 days maximum
- Gestation: Approximately 21 days
- Treatment End: On day 4 post partum
- Necropsy: On day 4 post partum (pups on day 4 post partum)

Remarks:

Doses / Concentrations:
0 (control), 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 males and 12 females per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

Dose levels were selected in agreement with the Sponsor, based on the results of a dose range-finding study

Maternal examinations:

VIABILITY / MORTALITY

Twice daily

CLINICAL SIGNS

Daily cage-side clinical observations were performed once daily during acclimatization period. Thereafter, up to the necropsy, daily clinical observation was performed immediately before dosing, up to 30 minutes post-dosing and one hour after dosing. An additional observation was made five hours after dosing during the normal working week. Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION

Pre-pairing period days 1 - 8 and 8 - 13; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and days 1 - 4 post partum.

No food consumption was recorded during the pairing period.

WATER CONSUMPTION

Monitored daily by visual inspection of water bottles.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy

DETAILED CLINICAL OBSERVATIONS

Detailed clinical observations were performed outside the home cage in all P generation animals. In all females of P generation, it was performed once prior to the first administration of the test item, 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

On day 3 post partum, relevant parameters were performed with 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, constitution, skin, pupil 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, rectal temperature.

Additionally, locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded.

Low beams count was reported at 10-minute intervals as well as the total activity over the measuring period.

CLINICAL LABORATORY INVESTIGATIONS

Blood samples were obtained at the end of the pre-pairing period from 5 females from each group. 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.

Hematology:
The following hematology parameters were determined:

Complete Blood Cell Count:

- 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
- Reticulocytes

Coagulation:

- Prothrombin time (= Thromboplastin time)
- Activated partial Thromboplastin time

Clinical Biochemistry:
The following clinical biochemistry parameters were determined:

- Glucose
- Urea
- Creatinine
- Bilirubin, total
- Cholesterol, total
- Triglycerides
- Aspartate aminotransferase
- Alanine aminotransferase
- Alkaline phosphatase
- Gamma-glutamyl-transferase
- Sodium
- Potassium
- Chloride
- Calcium
- Phosphorus
- Protein, total
- Albumin
- Globulin
- Albumin/Globulin ratio
- Bile acids

TERMINATION AND NECROPSY

Dams were sacrificed on day 4 post partum.

If birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

For the parent animals, special attention was directed at the organs of the reproductive system. 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, the following organs were trimmed from any adherent tissue, as appropriate, and their wet weight taken:

From all parental females:
- Ovaries (as pairs)
- Uterus and Cervix

From all parental animals of both genders:
- Adrenal glands (weighed as pairs)
- Brain
- Heart
- Kidneys (weighed as pairs)
- Liver
- Pituitary
- Thymus
- Spleen
- Thyroid

TISSUE PRESERVATION

Tissues from the following reproductive organs from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution:
- Ovaries
- Uterus
- Vagina
- Cervix

In addition, from all females at scheduled necropsy 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 (including cerebrum, cerebellum and pons)
- Spinal cord (cervical, mid-thoracic and lumbar)
- Small and large intestines (caecum, colon, duodenum, rectum, ileum and jejunum incl. Peyer’s patches)
- Stomach
- Liver
- Kidneys
- Adrenals
- Spleen
- Heart
- Thymus
- Thyroids
- Trachea and lungs (preserved by inflation with fixative and then immersion)
- Urinary bladder
- Lymph nodes (mesenterial, mandibular)
- Peripheral nerve (sciatic)
- Aorta (thoracic)
- Bone and bone marrow (femur including stifle joint)
- Bone and bone marrow (sternum)
- Muscle (skeletal)
- Oesophagus
- Parathyroid
- Pancreas
- Pituitary
- Salivary glands (sublingual / mandibular)
- Skin (abdominal region)
- Eyes (fixed in Davidson's fluid)
- Mammary tissue

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. Special stains were used at the discretion of the study pathologist.

HISTOPATHOLOGY

Slides of all organs and tissues collected at terminal sacrifice from the first five females which gave birth and slides of all reproductive organs from all females 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 and to all unfertile animals.

Histological examination of ovaries was carried out on any females that did not give birth.

A histopathology peer review was performed.

Ovaries and uterine content:

The ovaries and uterus were examined after termination on day 4 post partum. Examinations included the number of corpora lutea and the number of implantation sites.

Fetal examinations:

Not performed

Statistics:

The following statistical methods were used to analyze the grip strength, locomotor activity, food consumption, body weight, macroscopical findings, organ weights and ratios, clinical laboratory data, as well as reproduction data:

- 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.

Indices:

From the on-line recorded reproduction data, the following parameters were calculated: mean precoital time, percentage mating, fertility index, conception rate, pre and post-implantation loss, gestation index, birth index and viability index, dead/live pups at first litter check and pup sex ratio

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Mucosal parakeratosis of the vagina was observed in 3 out of 4 non pregnant females treated at 1000 mg/kg bw/day. A relationship with the test item treatment cannot be excluded.

Details on maternal toxic effects:
1. IN-LIFE DATA

VIABILITY / MORTALITY

No test item-related premature deaths were recorded.

Female no. 61 (100 mg/kg bw/day) was found dead on day 1 of the gestation period. An abnormal gait, sedation (grade 2 of 3), hunched posture, ruffled fur (grade 2 of 3), and tachypnea (grade 2 of 3) were observed before its premature death. The main microscopic finding was a marked diffuse acute inflammation in the lungs that was indicative of a gavage accident.

GENERAL CAGESIDE OBSERVATIONS (DAILY)

No test item-related clinical signs were found at any dose level.

A kinked tail apex, hair loss, vaginal bleeding and salivation were observed in individual animals and were considered to be incidental findings and not treatment-related.

DETAILED CLINICAL OBSERVATIONS (WEEKLY)

No test item-related clinical signs were found at any dose level.

A kinked tail apex was considered to be an incidental finding and not treatment-related.

FUNCTIONAL OBSERVATIONAL BATTERY

No test item-related effects on behavior, reflexes, grip strength, body temperature and locomotor activity were observed.

Statistically significant decreased body temperatures in all test item-treated female groups were considered to be caused by unusually high body temperatures in the control groups. Furthermore, the decrease was not dose-related and the values recorded in the test item-treated groups were within the range of the historical controls.

Statistically significant increased locomotor activity in females treated with 100 mg/kg bw/day was considered to be incidental, because the effect was not observed at higher dose levels.

FOOD CONSUMPTION OF FEMALES

No effects on food consumption of females were observed at any dose level.

BODY WEIGHTS OF FEMALES

No effects on body weight development in females were recorded.

2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

No effects on hematology parameters were recorded.

A change in a single parameter achieving individual statistical significance was considered to be unrelated to treatment, because it lacked a dose-relationship and was clearly within the historical control range.

CLINICAL BIOCHEMISTRY

No effects on biochemistry parameters were recorded.

Changes in several parameters achieving individual statistical significance were considered to be unrelated to treatment, because they lacked a dose-relationship and/or were clearly within the historical control range.

3. PATHOLOGY (PARENTAL FEMALES)

ORGAN WEIGHTS

No test-item related effects on organ weights were observed.

MACROSCOPIC FINDINGS

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

All findings were those commonly seen in the laboratory rat.

MICROSCOPIC FINDINGS

- Non Pregnant Females: Mucosal parakeratosis of the vagina was observed in 3 out of 4 non pregnant females treated with 1000 mg/kg bw/day. The change was characterized by a moderately thickened vacuolated epithelium showing early keratinization and desquamation of parakeratotic cells. In addition, the change was diffuse, more or less homogeneous, and involved areas away from the vaginal opening up to the cervix. The uterine endometrium was lined by tall columnar finely vacuolated cells while the ovary was unremarkable. This finding was considered to be of uncertain toxicological significance. In the remaining non pregnant animal of the 1000 mg/kg group, neutrophilic exudative inflammation was present in the uterus and was associated with suppurative exudate in the cervix and vagina; this was consistent with ascending bacterial infection, unrelated to the test item. There were no test item-related findings in any of the 3 or 1 non pregnant females treated with 100 and 300 mg/kg bw/day respectively.

- Pregnant Females: There were no test item-related microscopic findings, including following evaluation of the uterus or evaluation of follicles and corpora lutea in the ovaries.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: other:

Dose descriptor:

NOEL

Remarks:

(males)

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: other:

Dose descriptor:

NOEL

Remarks:

(females)

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Abnormalities:

not specified

Developmental effects observed:

not specified

1. REPRODUCTION, BREEDING AND PUP DATA

 

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litters

 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	49 - 60	61 - 72	73 - 84	85 - 96
Number of females paired	12	12	12	12
Number of females mated	12	12	12	12
Number of females, which died before the scheduled necropsy (A)	0	1	0	0
Numbers of females which did not deliver any pups (B)	0	3	2	4
Number of females which reared their pups until day 4post partum	12	8	10	7

 

(A) Female No. 61 died spontaneously on day 1post coitum

(B) Female Nos. 66, 68, 70, 76, 82 (had 2 implantation sites), 85, 87, 90, and 91

 

MATING PERFORMANCE AND FERTILITY

 

No test item-related effect on mating performance or fertility was observed at any dose level.

 

10, 10, 10, and 12 females mated in groups 1, 2, 3, and 4, respectively, within twelve days after initiation of pairing. Mean (median) precoital times were 2.3 (1), 2.5 (3), 4.2 (4) and 2.5 (3) days at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively. All six remaining females mated within 4 days of the second pairing period.

 

Eight females were not pregnant; four at the low dose level (female nos. 66, 68 and 70 mated with male nos. 13, 19, 20 and 22, respectively), one at the mid dose level (female no. 76 mated with male no. 27) and four at the high dose level (female nos. 85, 87, 90, and 91 mated with male nos. 37, 39, 42, and 43, respectively).

 

Consequently, fertility indices (number of females achieving pregnancy as a percentage of females paired) were 100%, 73%, 92% and 67% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 

The incidence of infertility did not follow a dose dependency and the number of non-pregnant females per group was in the range of the historical background, up to four non-pregnant females were noted in historical control groups. For these reasons lack of pregnancy was considered not to be related to the treatment with the test item.

 

Except for one female at 300 mg/kg bw/day all pregnant females gave birth to living pups. Consequently, the gestation index (number of females with living pups as a percentage of females pregnant) was 100%, 100%, 91%, and 100% in groups 1, 2, 3, and 4, respectively.

 

PRE-IMPLANTATION LOSS

 

No test item-related effects on pre-implantation loss were observed at any dose level.

 

Mean pre-implantation loss per dam was 1.3, 0.9, 1.1 and 2.0 at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 

CORPORA LUTEA COUNT

 

No effects on corpora lutea count were observed at any dose level.

 

Mean number of corpora lutea per dam was 14.7, 15.3, 15.5 and 14.4 at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 

DURATION OF GESTATION

 

No effects on duration of gestation were observed at any dose level.

 

Mean duration of gestation was 21.9, 21.5, 21.6 and 22.0 days at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 

IMPLANTATION RATE AND POST IMPLANTATION LOSS

 

No effects of the test item on implantation rate or post implantation loss were observed at any dose level.

 

Mean number of implantations per dam was 13.3, 14.4, 14.4 and 12.4 whereas mean number of post-implantation loss per dam was 1.5, 3.4, 1.4 and 2.5; both cited in order of ascending dose levels.

 

LITTER SIZE AT FIRST LITTER CHECK

 

No effects on litter size were observed at any dose level.

 

Mean numbers of living pups per dam at first litter check were 11.8, 11.0, 13.2 and 9.9, whereas birth indexes (number of pups borne alive as a percentage of implantations) were 88.8%, 76.5%, 91.7% and 79.8% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 

POSTNATAL LOSS DAYS 0 – 4 POST PARTUM

 

No effects on postnatal loss were observed at any dose level.

 

In the control group, one pup (from litter no. 52) was missing on day 3 post partum.

 

At the dose level of 1000 mg/kg bw/day, one pup (from litter no. 86) was found dead on day 1 post partum and one pup (from litter no. 94) was missing on day 1 post partum.

 

No further mortality of pups was observed at any dose level. Because the incidence of pups mortality did not correlate with the dose levels, the postnatal loss was considered not to be test item-related.

 

EXTERNAL EXAMINATION OF PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

No test item-related findings were noted in pups at any dose level.

 

PUP 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%, 45%, 47% and 53%, in order of ascending dose level.

 

RIGHTING REFLEX OF PUPS

 

No effects on righting reflex of pups were observed at any dose level.

 

Percentages of pups for which righting reflex was observed within the first one minute of the test were 86%, 84%, 83% and 96% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

 

BODY WEIGHTS OF PUPS TO DAY 4 POST PARTUM

 

No effects on pups body weights or body weight gain were noted at any dose level.

 

MACROSCOPIC FINDINGS OF PUPS

 

No findings were noted during the necropsy of pups at any dose level.

 

2. IN-LIFE DATA OF PARENTAL MALES

 

VIABILITY / MORTALITY

 

No test item-related premature deaths were recorded.

 

Male no. 4 (0 mg/kg bw/day, control group) was sacrificed on day 19 of the pairing period due to severe clinical signs which included muscular hypotonus (grade 3 of 3), decreased activity (grade 3 of 3), hunched posture, ruffled fur (grade 2 of 3), and reddish nasal secretion (grade 3 of 3). At necropsy, several reddish spots in the stomach mucosa were noted. Histopathologically no abnormalities were noted which could explain the cause of the moribund condition of the animal.

 

GENERAL CAGESIDE OBSERVATIONS (DAILY)

 

No test item-related clinical signs were found at any dose level.

 

A kinked tail apex, hair loss, and salivation were observed in individual animals and were considered to be incidental findings and not treatment-related.

 

DETAILED CLINICAL OBSERVATIONS (WEEKLY)

 

No test item-related clinical signs were found at any dose level.

 

A kinked tail apex was considered to be an incidental finding and not treatment-related.

 

FUNCTIONAL OBSERVATIONAL BATTERY

 

No test item-related effects on behavior, reflexes, grip strength, body temperature and locomotor activity were observed.

 

Statistically significant increased locomotor activity in males treated with 1000 mg/kg bw/day was considered to be incidental, because the effect was restricted to the first two 10 min intervals, not dose-related and no other findings (e.g. behavioral abnormalities) correlated with this effect.

 

FOOD CONSUMPTION OF MALES

 

No effects on food consumption of males were observed at any dose level.

 

BODY WEIGHTS OF MALES

 

No effects on body weight development in males were recorded.

 

3. CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL MALES

 

HEMATOLOGY

 

No effects on hematology parameters were recorded.

 

A change in a single parameter achieving individual statistical significance was considered to be unrelated to treatment, because it lacked a dose-relationship and was clearly within the historical control range.

 

CLINICAL BIOCHEMISTRY

 

No effects on biochemistry parameters were recorded.

 

Changes in several parameters achieving individual statistical significance were considered to be unrelated to treatment, because they lacked a dose-relationship and/or were clearly within the historical control range.

 

3. PATHOLOGY (PARENTAL MALES)

 

ORGAN WEIGHTS

 

No test-item related effects on organ weights were observed.

 

MACROSCOPIC FINDINGS

 

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

 

Reduced size of testes/epididymides in 1/12 males (no.37) recorded at 1000 mg/kg bw/day is a common incidental finding in rats of this age and strain.

 

All other findings were those commonly seen in the laboratory rat.

 

MICROSCOPIC FINDINGS

 

There were no test item-related findings in any examined organs including reproductive organs. In particular, the qualitative examination of the stages of spermatogenesis in the testis did not reveal any test item-related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle. The reduced size of testes/epididymides observed in rat no.37 (1000 mg/kg bw/day) was correlated histologically with moderate multifocal testicular germ cell degeneration leading to moderate abnormal germinal cell content in the epididymides. In the absence of any test-item related effects on male reproductive organs (organ weights or other microscopic change) this isolated finding, known to occur spontaneously in rat, was considered to be fortuitous.

Conclusions:

The NOEL (No Observed Effect Level) for general toxicity in males and for reproduction/developmental toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest dose level used. Due to finding of mucosal parakeratosis of the vagina in females at 1000 mg/kg bw/day, the NOEL for general toxicity in females was considered to be 300 mg/kg bw/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test tiem 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.

 

The test item was administered to male rats for 46 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 for a period of 40 to 56 days.

 

The following dose levels were applied:

 

Group 1:      0 mg/kg body weight/day (control group)

Group 2:  100 mg/kg body weight/day

Group 3:  300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (Arachis oil).

 

The following results were obtained:

PARENTAL ANIMALS:

 

MORTALITY AND GENERAL TOLERABILITY

 

No test item-related premature deaths were recorded.

 

No test item-related findings were noted during daily clinical or detailed weekly clinical observations in males or females at any dose level.

 

FUNCTIONAL OBSERVATIONAL BATTERY

 

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

 

FOOD CONSUMPTION

 

No effects on food consumption of males or females were observed at any dose level.

 

BODY WEIGHTS

 

No effects on body weights or body weight gain of males or females were observed at any dose level.

 

CLINICAL LABORATORY INVESTIGATIONS

 

No test item-related effects on hematology or clinical biochemistry parameters were noted in males or females at any dose level.

 

REPRODUCTION AND BREEDING DATA

 

No test item-related effect on mating performance, fertility, duration of gestation, corpora lutea count, pre-implantation loss, implantation rate, post implantation and postnatal loss or litter size were noted at any dose level.

 

ORGAN WEIGHTS

 

No test item-related effects on absolute organ weights or organ weights relative to body or brain weights were noted in males or females at any dose level.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATIONS

 

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

 

There were no test item-related findings in any examined organs including reproductive organs in all males and all pregnant females.

Mucosal parakeratosis of the vagina was observed in 3 out of 4 non pregnant females treated with 1000 mg/kg bw/day. This change was diffuse, more or less homogeneous, and involved areas away from the vaginal opening up to the cervix. Although no other findings correlated with the parakeratosis, a relationship with the test item treatment cannot be excluded. There were no test item-related findings in any of the 3 non pregnant females treated with 100 mg/kg bw/day or the 1 non pregnant female treated with 300 mg/kg bw/day.

LITTER DATA - FI PUPS:

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

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

 

No effects on righting reflex were observed in pups at any dose level.

 

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

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 

No effects on pups body weights or body weight gain were noted at any dose level.

 

MACROSCPICAL FINDINGS IN PUPS

 

No findings were noted during the necropsy of pups at any dose level.

 

 

CONCLUSION

 

Based on these results, the NOEL (No Observed Effect Level) for general toxicity in males and for reproduction/developmental toxicity in males and females was considered to be 1000 mg/kg bw/day, the highest dose level used. Due to finding of mucosal parakeratosis of the vagina in females at 1000 mg/kg bw/day, the NOEL for general toxicity in females was considered to be 300 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study has been conducted according to OECD Guideline 422 and GLP and is adequately reported. The study has been assigned a reliability of 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Han Wistar Rat

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item to rats. The test item was administered in Arachis oil as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. The test item was administered to male rats for 46 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.

Development was not affected by the treatment. There were no test item-related findings in pups noted during the first litter check, the first 4 days post partum or during the necropsy. Pups body weights and body weight gain were not affected at any dose level.

The NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level used.

Justification for selection of Effect on developmental toxicity: via oral route:
The available OECD 422 study is assigned as reliability study 1 and is the only reproductive/developmental toxicity study available.

Justification for classification or non-classification

Reproduction and development were not affected by the treatment at any dose level, including the highest dose level of 1000 mg/kg bw/day. Mating performance, fertility, duration of gestation, corpora lutea count, implantation rate, post implantation and postnatal loss or litter size were similar in the control and all dose groups. There were no test item-related findings in pups noted during the first litter check, the first 4 days post partum or during the necropsy. Pups body weights and body weight gain were not affected at any dose level.

Therefore, based on the results of the OECD 422 study there is no evidence that the substance has an adverse effect on sexual function and fertility, or on development at the highest dose level. The substance is therefore not classified for reproductive toxicity based on this study.