Reaction mass of hexadecyl dihydrogen phosphate and dihexadecyl hydrogen phosphate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

12-Jul-2012 to 24-Jan-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP and OECD guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hexadecyl dihydrogen phosphate

EC Number:

222-581-1

EC Name:

Hexadecyl dihydrogen phosphate

Cas Number:

3539-43-3

Molecular formula:

C16H35O4P

IUPAC Name:

hexadecyl dihydrogen phosphate

Test material form:

other: whithe powder

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system 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): 302 to 366 g (males) and 193 to 219 g (females)
- Identification: Cage card and individual animal number (ear tattoo).
- 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: 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 (The temperature range was 20.3 - 26.9 °C; relative humidity range was: 40.8 - 90.7%). Values outside of these ranges occasionally occurred, usually following room cleaning, which was considered not to have any influence on the study. There was 12-hour fluorescent light / 12-hour dark cycle with music during the light period.
- Accommodation: In groups of three to five animals in Makrolon type-4 cages with wire mesh tops up to the day of ran-domization 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. 0502, 0601, 0201 and 6960C.CS-100099. 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. Bacteriological assay, chemical and contaminant analyses of representative samples were performed.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: highly purified water (ELGA)

Details on exposure:

DOSE FORMULATIONS

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

The appropriate amount of the test item was weighed into a speed mixer beaker or an appropriate weighing pan on a tared precision balance. A certain amount of vehicle (highly purified water) was added to the test item and the beaker were placed in the speed mixer (DAC 150.1 FVZ) and mixed at 3000 rpm for at least 5 minutes until a homogeneous suspension was prepared. Separate formulations were prepared for each concentration.

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

STORAGE OF DOSE FORMULATIONS

Based upon the results of stability analyses performed within the Harlan Laboratories non-GLP study D51812, dose formulations were stable for at least 4 hours at room temperature (15 - 25 °C) and for 8 days in refrigerator (2 - 8 °C).

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Target Dose Levels: 0 mg/kg/day (Group 1, control group), 100 mg/kg/day (Group 2), 300 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 D51812, using dose levels of 0, 100, 300 and 1000 mg/kg/day.
- Dose Volume: 10 mL/kg body weight
- Dose Concentrations: 0 mg/mL (Group 1), 10 mg/mL (Group 2), 30 mg/mL (Group 3) and 100 mg/mL (Group 4)
- Duration of Acclimatization Period: Minimum 5 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

METHODS

On the first treatment day, on 19-Jul-2012, 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 to confirm the stability (4 hrs at room temperature). On 10-Aug-2012 samples were taken from the leftover dose preparation of group 4 from the middle to confirm concentration. 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 responsible person for formulation analysis (Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to a ELSD detector following an analytical procedure developed in Harlan Laboratories Switzerland. The test item was used as the analytical standard. Analyzed samples were not discarded without written consent from the study director.

Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were not discarded without written consent from the study director.

RESULTS

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 the 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 the test item in the range of 92.2% to 113.1% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of the test item in the preparations was approved because single results found did not deviate more than 2.9% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept four hours 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 approved.

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 commenced.

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:

Males: Minimum 4 weeks
Females: Approximately 7 weeks

Frequency of treatment:

Once daily

Duration of test:

MALES

- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Treatment Ends: On day before sacrifice
- Necropsy: After a minimum of 28 days treatment. Sperm analysis performed in 10 males per group and blood sampling on the day of necropsy performed in 5 males per group

FEMALES

- Acclimatization: 5 days minimum
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 4 post partum
- Necropsy: On day 4 post partum (pups) or on day 5 post partum (dams). Blood sampling performed in dams on day 5 post partum

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 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 the test item on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition. The test item was administered once daily orally (by gavage) to male and female rats throughout the pre-pairing and pairing periods, after pairing (males), gestation and lactation periods (females) including the day before scheduled necropsy. Sperm analysis of selected males was performed.

Examinations

Maternal 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

Pre-Pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum.

No food consumption was recorded during the pairing period.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy.

CLINICAL LABORATORY INVESTIGATIONS

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 assay was performed at Harlan Laboratories Ltd. (Füllinsdorf) under internal laboratory quality control conditions to assure reliable test results.

- First Blood Sampling of Females: on Day 5 Post Partum on 28-Aug-2012

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

Coagulation:

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

The following clinical biochemistry parameters were determined:

- Calcium
- Phosporus

TERMINATION AND NECROPSY

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 animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes at the scheduled necropsy. 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, organs were weighed as listed below. Organs were trimmed from any adherent tissue, and their wet weight taken. Adrenals and kidneys were weighed separately.

- Kidneys for all animals
- Thymus
- Adrenals

TISSUE PRESERVATION

Unless indicated otherwise, the following organs were preserved in neutral phosphate buffered 4% formaldehyde solution.

- Ovaries
- Uterus, Vagina, Cervix
- Kidneys for all animals
- Lungs (inflated approximately with 4% formalin before immersion in fixative)
- Thymus
- Adrenals
- Ileum, with Peyer's patches
- Jejunum with Peyer's patches
- Stomach
- Duodenum
- Caecum
- Colon
- Rectum
- Gross Lesions

HISTOTECHNIQUE

All organ and tissue samples to be examined by the principal investigator 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 animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions and to all animals, which died spontaneously or had to be terminated in extremis.

If test item-related morphologic changes were detected in organs of any high-dose animal, those same organs from the mid- and low-dose groups 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.

A histopathology peer review was performed and the results were included in the histopathology phase report.

Ovaries and uterine content:

The ovaries and uterus were examined after termination on day 5 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 food consumption, body weights 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.

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.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

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

VIABILITY/MORTALITY

All animals survived the scheduled study period.

CLINICAL SIGNS OR OBSERVATIONS

No test item-related clinical signs were observed during the study.

Breathing noises were recorded in some females (no. 56 in group 2 and nos. 79, 82, 85, 86 and 88 in group 4) in the gestation period for a short time period (from 1 to 5 days). These clinical signs were related to the method of application which changed during the study from microlab technique to standard gavage application and to the fact that the dose formulation of the high dose was very thick and therefore not easily tolerated when administered to some animals.

Hairloss was noted for a few females during the study across the dose groups but this finding was considered to be incidental.

No further clinical signs were noted in females at any dose level.

FOOD CONSUMPTION OF FEMALES

Food consumption of females was not affected by the treatment with the test item.

BODY WEIGHTS OF FEMALES

No test item-related effects on body weights and body weight gain of females were observed at any dose level.

2. CLINICAL LABORATORY INVESTIGATIONS

HEMATOLOGY

No test item-related effects were found on hemathology parameters in females.

CLINICAL BIOCHEMISTRY

No test item-related effects were found on clinical biochemistry parameters in females.

3. TERMINAL FINDINGS

ORGAN WEIGHTS

No test item-related effects on measured organ weights of females were noted.

MACROSCOPICAL FINDINGS

No test item-related findings were noted during necropsy of females at any dose level. Findings that occurred in females were considered to be within the range of normal background alterations: foci in stomach, lungs or thymus, dilated uterus and watery cyst on ovaries were incidentally found.

HISTOPATHOLOGY FINDINGS

Under the conditions of this experiment HOSTAPHAT CC 100 (Hexadecyl dihydrogen phosphate), at the end of the treatment period did not cause neither macroscopic nor microscopic treatment-related changes.

The examination of the ovaries did not reveal any morphological difference between control and treated animals. The qualitative assessment of the male genital organs did not reveal any treatment-related effect.

The ovaries of females that did not give birth did not present any pathological change which might have explained the infertility. The corresponding mating males were also devoid of any pathological change in the genital organs, therefore based on the organ morphology an explanation to the infertility was not possible to attain. However, as there was not a dose-relationship (high dose animals were not affected) the infertility in these animals was unrelated to the treatment with the test item.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: developmental toxicity

3

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Basis for effect level:

other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:not examined

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

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	45 - 55	56 - 66	67 - 77	78 - 88
Number of females paired	11	11	11	11
Number of females mated (A)	11	11	11	11
Number of pregnant females	9	7	10	10
Number of non-pregnant females (B)	2	4	1	1
Number of females, which died before the scheduled necropsy	0	0	0	0
Number of females, which lost their litters	0	0	0	0
Numbers of females,which did not deliver any pups	0	0	0	0
Number of females which reared their pups until day 4 post partum	9	7	10	10

 

(A) For female no. 60 in group 2 and for females nos. 69 and 77 in group 3 mating was not detected but they were pregnant

(B) Female nos. 46 and 52 in group 1, females 56, 57, 64, 66 in group 2, female no. 68 in group 3 and female no. 86 in group 4 were not pregnant.

 

MATING PERFORMANCE AND FERTILITY

 

No effects on mating performance or fertility were observed at any dose level.

 

With exception for one female in the control group (no. 55) mating of all females was recorded during the first pairing period. The precoital time was not affected by the treatment with the test item. Mean precoital times calculated for the first pairing period were 2.2, 3.1, 2.7 and 2.8 days in order of ascending dose levels.

 

Mating of female no. 60 in group 2 and nos. 69 and 77 was not detected during the first pairing period and these females were paired with a second partner for a second pairing period. Body weight gain of these three females recorded during the first days of the second pairing period increased indicating a pregnancy. For this reason the second pairing period was interrupted. Both females gave birth which confirmed that their mating during the first pairing period was overlooked. 

 

Two females in the control group (nos. 46 and 52), four females in group 2 (56, 57, 64, 66), one female each in group 3 (no. 68) and group 4 (no. 86) were not pregnant. Fertility indexes (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 81.8% in the control group, 63.6 in group 2 and 90.9 in groups 3 and 4. Except for group 2 where the fertility rate was exceptionally low the values were within the historical control range, and due to the lack of dose dependency any relation to the treatment with the test item can be excluded.

 

All remaining females gave birth to living pups. Gestation index (number of females with living pups as a percentage of females pregnant) was 100% in all groups.

 

DURATION OF GESTATION

 

No effects on duration of gestation were observed at any dose level. The mean values of test item-treated groups were similar to the control.

 

CORPORA LUTEA COUNT

 

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

 

IMPLANTATION RATE AND POST IMPLANTATION LOSS

 

No effects on implantation rate and post-implantation loss were noted.

 

The overall number of implantations per dam was 12.1, 12.4, 12.3 and 12.1 in order of ascending dose level. The overall mean number of post-implantation loss per dam was 0.8, 1.0, 1.2 and 1.3 at the dose level of 0, 100, 300 and 1000 mg/kg body weight/day and the values were within the historical background range.

 

LITTER SIZE AT FIRST LITTER CHECK

 

At the dose level of 1000 mg/kg bw/day, consequent to the slightly higher post-implantation loss a lower litter size was recorded at first litter check; mean number of living pups per dam was 10.8 compared to 11.3 in the control group. This resulted in birth index (number of pups borne alive as a percentage of implantations) of 89.3% at the high dose compared to 93.6% in the control group. The differences were not statistically significant. Mean value of living pups per dam at first litter check was in the range of the historical controls containing values from 10.3 to 13.2. Therefore, test item-related effect on the litter size at the high-dose level can be excluded.

 

At the dose levels of 100 and 300 mg/kg bw/day, no effects on litter size were noted.

 

The overall mean numbers of living pups per dam at first litter check were 11.3, 11.4, 11.1 and 10.8, whereas birth indexes (number of pups borne alive as a percentage of implantations) were 93.6%, 92.0%, 90.2% and 89.3% at the dose levels of 0, 100, 300 and 1000 mg/kg body weight/day, respectively.

 

Two dead pups in one litter were found in group 4, but this was within the historical background range, therefore it was considered not to be related to the treatment with the test item.

 

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM

 

No effect on post natal loss up to Day 4 post partum was observed in any dose groups.

 

The mean postnatal loss per dam between days 0 and 4 post partum was 0.1, 0.0, 0.1 and 0.0 in ascending dose levels.

 

EXTERNAL EXAMINATION OF PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

No test item-related observations were noted in pups during the first litter check or up to day 4 of lactation 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 55%, 48%, 45% and 47%, in order of ascending dose level.

 

BODY WEIGHTS OF PUPS TO DAY 4 POST PARTUM

 

No effects on pup body weights were noted at any dose level.

 

Mean body weights of pups on day 1 post partum were: 6.2 g, 6.1 g, 6.7 g and 6.4 g, at the dose levels of 0, 100, 300 and 1000 mg/kg/day respectively, body weight gain of pups during the first four days of the lactation period was +46.8%, +43.0%, +39.6% and +46.3%, respectively.

 

MACROSCOPICAL FINDINGS OF PUPS

 

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

 

2. IN-LIFE DATA OF PARENTAL MALES

 

VIABILITY/MORTALITY

 

All animals survived the scheduled study period.

 

CLINICAL SIGNS OR OBSERVATIONS

 

No test item-related clinical signs were observed during the study.

 

Breathing noises were recorded in some males (no. 32 in group 3, nos. 34, 36, 37, 38, 40, 41, 44 in group 4) during the pairing for a short time period (from 1 to 5 days). These clinical signs were related to the method of application which changed during the study from microlab technique to standard gavage application and to the fact that the dose formulation of the high dose was very thick and therefore not easily tolerated when administered to some animals.

 

No further clinical signs were noted in males at any dose level.

 

FOOD CONSUMPTION OF MALES

 

No effect on food consumption was noted in males in any dose group.

 

BODY WEIGHTS OF MALES

 

At the dose level of 1000 mg/kg bw/day, statistically significantly increased body weight gains were noted in males during the pre-pairing period (except for days 2, 6, 7, 13). This was considered unlikely to be test item-related since difference was less than 5% from controls and no effects were observed on absolute body weights.

 

At 300 mg/kg bw/day, the body weights of males was statistically significantly decreased on day 19 of the pairing period but this was considered to be of no toxicological significance.

 

3. CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL MALES

 

HEMATOLOGY

 

No test item-related effects were found on hemathology parameters in males.

 

At 1000 mg/kg body weight/day, the mean corpuscular hemoglobin concentration in males was statistically significantly higher when compared to controls but the value was within the historical control data. This difference was noticed due to one animal (no. 35) in this dose group.

The hemoglobin concentration distribution width was statistically significantly decreased in males in the high dose group but the value (1.54) was just below the historical control data (1.55-2.14). Statistifically significantly higher eosinophil counts were also noted in group 4 males but the value was within the historical control data. Therefore, these changes were considered to be of no toxicological importance.

 

In groups 3 and 2 no test item-related changes were noted.

 

CLINICAL BIOCHEMISTRY

 

No test item-related effects were found on clinical biochemistry parameters in males.

 

At 1000 mg/kg body weight/day, the calcium level of males was statistically significantly lower when compared to controls but the value (2.66 mmol/l) was within the historical control data (2.51 - 2.91 mmol/l), therefore this was not considered to be treatment-related effect.

 

No changes were observed in groups 3 and 2.

 

4. TERMINAL FINDINGS IN PARENTAL MALES

 

SEMINOLOGY AND SPERMATID COUNT

 

- Sperm Motility: sperm motility was not affected by treatment with the test item at any dose level. Motility scores for groups 2 - 4 were similar to those of the control group.

 

- Morphology: the morphology of the spermatids was not affected by treatment with the test item. The percentage of complete spermatide with normal hook and tail was 96.8% in group 4, compared to 96.7% in the control group.

 

- Spermatid Head Count: determination of homogenization-resistant spermatids obtained from cauda epididymis or testes tissue samples gave no indication for any test item-related effect (examination was performed for groups 1 and 4 only). Per gram of epididymidal tissue 740.5 Mio spermatids were counted in group 4, compared to 763.0 Mio spermatids in the control group. Per gram of testis tissue 116.0 Mio spermatids were counted in group 4, compared to 124.9 Mio spermatids in the control group.

 

ORGAN WEIGHTS

 

At the dose level of 1000 mg/kg bw/day, the absolute and relative thymus weights of males were statistically significantly lower when compared to controls but the values were within the historical control range. Additionally there were no histopathological changes observed in the thymus, therefore this was considered to be incidental.

 

No further changes of organ weights were noted at any dose level.

 

MACROSCOPICAL FINDINGS

 

no test item-related findings were noted at any dose level. Testes and epididymides were reduced in size in one male each in group 2 (no.17) and in group 4 (no. 42). The male in group 2 was fertile but the partner of group 4 male was not pregnant. Since these findings occurred in other studies in this laboratory this was considered to be incidental. The remaining findings such as pelvic dilation of kidneys, foci in lungs discolouration of lymph nodes or Peyer’s patches occurred sporadically across the dose groups and were considered not to be related with the treatment with the test item.

 

HISTOPATHOLOGY FINDINGS

 

Under the conditions of this experiment HOSTAPHAT CC 100 (Hexadecyl dihydrogen phosphate), at the end of the treatment period did not cause neither macroscopic nor microscopic treatment-related changes.

 

The ovaries of females that did not give birth did not present any pathological change which might have explained the infertility. The corresponding mating males were also devoid of any pathological change in the genital organs, therefore based on the organ morphology an explanation to the infertility was not possible to attain. However, as there was not a dose-relationship (high dose animals were not affected) the infertility in these animals was unrelated to the treatment with the test item.

Applicant's summary and conclusion

Conclusions:

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 highly purified water 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 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.

At the dose level of 1000 mg/kg bw/day, statistically significantly increased body weight gains were noted in males during the pre-pairing period. This was considered to be incidental. The absolute and relative thymus weights of males were statistically significantly lower when compared to controls but the values were within the historical control range. Additionally there were no histopathological changes observed in the thymus, therefore this was considered to be incidental.

Macroscopical and microscopical examination of males and females did not reveal any test item-related effect.

No effects on mating performance, fertility, corpora lutea count, duration of gestation, post-implantation loss, litter size or breeding loss were observed at any dose level.

No test item-related findings were noted at first litter check and during lactation in pups at any dose level.

Based on these results, NOEL (No Observed Effect Level) for general toxicity in males and females was considered to be 1000 mg/kg body weight/day.

The NOEL for reproduction/developmental toxicity was considered to be 1000 mg/kg body weight/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of the test item on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

 

Four groups of 11 males and 11 females were treated by gavage with the test item once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum.

 

The following dose levels were used:

 

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 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water (ELGA)).

 

The following results were obtained:

 

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 

All animals survived the scheduled study period.

 

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

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 

Food consumption was not affected by the treatment with the test item at any dose level.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 

Body weights and body weight gains of males and females were not affected by the treatment up to and including the dose level of 1000 mg/kg body weight/day.

 

REPRODUCTION AND BREEDING DATA

 

No effects on mating performance, fertility, corpora lutea count, duration of gestation, post-implantation loss, litter size or breeding loss were observed at any dose level.

 

CLINICAL LABORATORY INVESTIGATIONS IN PARENTAL ANIMALS

 

No test item-related effects were found on hemathology or biochemistry parameters in males or females.

 

ORGAN WEIGHTS OF PARENTAL ANIMALS

 

At 1000 mg/kg body weight/day, there was a statistically significantly reduced thymus weight in males measured which was considered to be incidental since it was within the historical background range.

 

SPERM ANALYSES IN PARENTAL MALES

 

Sperm analysis did not reveal any test item-related effects.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATION OF PARENTAL ANIMALS

 

Macroscopical and microscopical examinations did not reveal any test item-related effects.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK

 

No test item-related findings were noted 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 pup body weights or body weight gain were noted at any dose level.

 

MACROSCOPICAL FINDINGS IN PUPS

 

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

 

CONCLUSION

 

Based on these results, NOEL (No Observed Effect Level) for general toxicity in males and females was considered to be 1000 mg/kg body weight/day.

 

The NOEL for reproduction/developmental toxicity was considered to be 1000 mg/kg body weight/day.