Reaction mass of Dimethyl [3-[(hydroxymethyl) amino]-3-oxopropyl] phosphonate and Dimethyl (3-amino-3-oxopropyl)phosphonate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl [3-[(hydroxymethyl)amino]-3-oxopropyl]phosphonate

EC Number:

243-528-9

EC Name:

Dimethyl [3-[(hydroxymethyl)amino]-3-oxopropyl]phosphonate

Cas Number:

20120-33-6

Molecular formula:

C6H14NO5P

IUPAC Name:

dimethyl [3-[(hydroxymethyl)amino]-3-oxopropyl]phosphonate

Constituent 2

Reference substance name:

Dimethyl (3-amino-3-oxopropyl)phosphonate

EC Number:

219-765-9

EC Name:

Dimethyl (3-amino-3-oxopropyl)phosphonate

Cas Number:

2526-69-4

Molecular formula:

C5H12NO4P

IUPAC Name:

dimethyl (3-amino-3-oxopropyl)phosphonate

Test material form:

other: liquid

Specific details on test material used for the study:

Name: DMPPA_701-402-5
Batch No: 0015273200
Colour: Colourless to yellowish
Expiry Date: 15 August 2012
Purity: ~ 70 %
Storage: Room temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals will be non-pregnant and nulliparous.
Age at the start of the treatment period: 9-10 weeks old
Body weight at the allocation of the animals to the experimental groups: males: 324–376 g. (mean: 353.50 g ± 20 % = 424.20 – 282.80 g), females: 172–214 g (mean: 197.1 g = 236.52 ± 157.68 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 0815)
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals will be kept individually in IVC cages (except during the mating period when two females will be paired with one male), type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 060411)
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days)


IN-LIFE DATES: From: 28 Dec 2011 To: 10 Feb 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

aqua ad injectionem (sterile water)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 20 mg/mL, 60 mg/mL, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
- Manufacturer: AlleMan Pharma
- Batch No: 110147
- Expiry Date: 02/2014
- Purity: 100 %

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: maximal 14 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for the nominal concentration verification were taken in study week 1 (first week of pre mating period), study week 3 (first week of mating), study week 5 (gestation) and study week 7 (gestation/lactation) freshly prepared low, medium, and high dose groups (12 samples in total). Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation in study week 1 and 5 (12 samples in total). Samples for stability analysis were taken from top and bottom level of high dose and low dose group in study week 1 at 0 hr and 6 hrs (8 samples in total). All concentration samples were stored frozen (approximately -20 °C) until the analysis was performed. The determination of test item concentration in the dosing formulations were performed by the analytics department of BSL Bioservice in accordance with GLP.

Duration of treatment / exposure:

The test item was administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days has been completed.

Frequency of treatment:

Daily

Details on study schedule:

After 14 days of treatment to both male and female, animals were paired (1:1) for maximum 14 days. The subsequent morning onwards, vaginal smears of the females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2 : Low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3: Mid dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4: High dose

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose level was chosen with the aim of inducing toxic effects, but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dose-related response and a NOAEL.

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once a day

BODY WEIGHT: Yes
- Time schedule for examinations: In the male animals, the body weight was taken weekly during the entire study period.
In the female animals, the body weight was taken weekly during the pre-mating period, on gestation day 0, 7, 14, 20 and on post-natal day 0 (within 24 hours of parturition) and post-natal day 4 along with pups.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Litter observations:

The duration of gestation was recorded and was calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed. Litters were weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing actual numbers on the back with the help of permanent marker. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Males were sacrificed any time after the completion of the mating period (minimum total dosing of 28 days).
- Maternal animals: Females were sacrificed on post-natum day 4

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
A histopathological evaluation (after the preparation of paraffin sections and haematoxylin-eosin staining) was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. A detailed qualitative examination of the testes was made taking into account the tubular stages of the spermatogenic cycle at the evaluation of additional haematoxylin-PAS (Periodic Acid Schiff) stained slides.

Postmortem examinations (offspring):

Dead pups and pups killed on day 4 post-partum or shortly thereafter were carefully examined externally for gross abnormalities.

Statistics:

A statistical assessment of the results of the body weight, food consumption and absolute and relative organ weights was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test.

Reproductive indices:

Copulation Index, Fertility Index, Delivery Index, and Viability Index were assessed.

Offspring viability indices:

The duration of gestation was recorded and was calculated from day 0 of pregnancy. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed. Litters were weighed within 24 hours of parturition (day 0 post-partum) and on day 4 post-partum.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were few clinical signs observed in male and female treatment groups. The clinical signs recorded in males were nasal discharge in an individual MD animal and aggressive behaviour in 2/10 LD and 2/10 MD animals. The clinical findings recorded in female animals were alopecia in 1/10 LD animals, slight piloerection in 1/10 LD animals, dehydration in 1/10 MD animals and a wound in 1/10 HD animals. Note, the dehydration occurred at a single day due to an accidentally withholding of water. The above clinical findings were not relevant to treatment. There was no mortality or morbidity recorded in C and treatment groups during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males, statistical analysis of body weight data revealed a significantly decreased body weight gain in MD animals between day 21-28 (mating/post-mating period) when compared to the C animals. However, evaluation of all other time points and groups revealed no considerable change. Hence, the significant change within MD animals is considered to be incidental and not test item related. DMPPA_701-402-5 did not have any effect on body weight development of female animals in this study. Throughout the treatment period body weights of female animals of all groups were in the normal range of variation for this strain and age. There were no considerable differences in body weight between any of the dose groups and the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

DMPPA_701-402-5 did not have any significant effect on food consumption of male and female animals in this study. A slight dose dependent decreased food consumption in the male treatment groups during pre-mating days 7-14 when compared to the C animals appeared due to a high food consumption of one individual animal of the C group (No. 8). The food consumption of all other male group C animals were comparable to the male animals of the treatment groups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No test item-related histological findings were noted in the male and in the female reproductive organs.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no treatment related difference observed for copulation index (%), fertility index (%), delivery index (%) and viability index (%) when compared to the control. Only one value (fertility index of LD animals) was below 100 % which results due to an individual animal which was not pregnant although a sperm positive smear was ascertained.

LITTER WEIGHT DATA:
DMPPA_701-402-5 did not have any effect on litter weight data in this study – neither on mean litter weight or total litter weight on day 0 and 4 nor on the number of male and female pups. On PND 0 and 4 litter weight data of all groups were in the normal range of variation. There were no considerable differences in litter weight between any of the dose groups and the control group.

PRECOITAL INTERVAL AND DURATION OF GESTATION: DMPPA_701-402-5 did not have any influence on precoital interval or duration of gestation in this study. All groups were in the normal range of variation for this strain and age. There were no considerable differences in precoital interval or duration of gestation between any of the dose groups and the control group.

PRE AND POST NATAL DATA: There was no statistically significant influence on number of corpora lutea, implantation sites, live pups born on PND 0 as well as the percentages of pre implantation loss and post implantation loss after comparing the treatment groups with the C group. A tendency to an increase in the percentage of post implantation loss could be detected for LD, MD, and HD groups. However, no dose dependency as well as a high variability among the individual animals was detected. Hence, the toxicological relevance remains unclear.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Reproductive toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

Litter Weight Data
For a detailed description of the individual findings see Table 14 and Appendix 9 of the report. DMPPA did not have any effect on litter weight data in this study – neither on mean litter weight or total litter weight on day 0 and 4 nor on the number of male and female pups. On PND 0 and 4 litter weight data of all groups were in the normal range of variation. There were no considerable differences in litter weight between any of the dose groups and the control group.

Precoital Interval and Duration of Gestation
For a detailed description of the individual findings see Table 15 and Appendix 10 of the report. DMPPA did not have any influence on precoital interval or duration of gestation in this study. All groups were in the normal range of variation for this strain and age. There were no considerable differences in precoital interval or duration of gestation between any of the dose groups and the control group.

Pre and Post Natal Data
For a detailed description of the individual findings see Table 16 and Appendix 11 of the report. There was no statistically significant influence on number of corpora lutea, implantation sites, live pups born on PND 0 as well as the percentages of pre implantation loss and post implantation loss after comparing the treatment groups with the C group. A tendency to an increase in the percentage of post implantation loss could be detected for LD, MD, and HD groups. However, no dose dependency as well as a high variability among the individual animals was detected. Hence, the toxicological relevance remains unclear.

Litter Data
For a detailed description of the individual findings see Table 17 and Appendix 12 of the report. There was no statistically significant difference observed for total number of born pups, number of males and females including sex ratio, live pups, still births, and runts at PND 0. Furthermore, no statistical significant difference was found for number of males and females including sex ratio as well as total number of live pups at PND 4. A tendency to a decreased sex ratio (m/f) was found in LD animals. However, this finding was not observed in MD and HD animals. Hence, it is assumed to be incidental.

Reproductive Indices
For a detailed description of the individual findings see Table 18 and Appendix 13 of the report. There was no treatment related difference observed for copulation index (%), fertility index (%), delivery index (%) and viability index (%) when compared to the control. Only one value (fertility index of LD animals) was below 100 % which results due to an individual animal which was not pregnant although a sperm positive smear was ascertained.

Pup Survival Data
For a detailed description of the individual findings see Table 19 and Appendix 14 of the report. One individual fetus of animal No. 75 (HD group) was found dead on PND 1. The viability index of this individual animal was 93.33 %. All other foetuses from this animal and the foetuses from all other animals survived till PND4. Hence, a viability index of 100% is given. Since only a single fetus from an individual animal was affected, this finding is assumed to be incidental.

Pup External Findings
For a detailed description of the individual findings see Appendix 17 of the report. There were two individual external findings recorded in foetuses of C and MD groups, which are as follows: Animal No. 49 (C) – one individual pup missed a piece of the tail. Animal No. 61 (MD) – one fetus exhibited a wound at the navel. There was no dose response pattern observed for the findings and were considered to be incidental.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

ca. 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related changes could be evaluated for the fetuses – neither for reproductive toxicology nor for developmental toxicology

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

The male Wistar rats after being treated with DMPPA_701-402-5 in aqua ad injectionem for the period of 28 days at the dosage of 100, 300 and 1000 mg/ kg body weight revealed no clear treatment related changes observed for the measured parameters when compared to the corresponding controls. Hence, the no observed adverse effect level (NOAEL) for male adult animals is assumed to be 1000 mg/kg body weight. The female rats were dose administered for approximately 54 days at the same dosage. There was no treatment related changes observed for any of the parameters when compared to the control. Hence, the no observed adverse effect level (NOAEL) for female adult animals is assumed to be 1000 mg/kg body weight. No treatment related changes could be evaluated for the fetuses – neither for reproductive toxicology nor for developmental toxicology. Hence, the no observed adverse effect level (NOAEL) for reproductive toxicology as well as for developmental toxicology is believed to be 1000 mg/kg body weight.

Executive summary:

The aim of this study was to assess the possible effect of DMPPA_701-402-5 on male, female fertility and embryofoetal development in Wistar rats. This study was conducted in accordance with OECD TG 421, OPPTS 870.3550 and Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, May 30, 2008. In this study, four groups comprising 10 adult male and 10 non pregnant nulliparous female rats (Wistar Crl:WI) were dosed daily by oral gavage with100, 300 and 1000 mg/kg body weight per day of DMPPA_701-402-5 at dose volume of 5 mL/kg body weight. The test item was formulated in aqua ad injectionem (sterile water) with administration volume of 5 mL/kg body weight. Control animals were handled identically as treated groups and received aqua ad injectionem (sterile water) in similar volume as treated groups. The test item formulation was prepared freshly and administered daily during 14 days pre mating and 14 days mating period in both male and in female, during gestation period and up to post natal day 3 in females. Males were dosed for 28 days. Dose volumes were adjusted based on the most recent body weight measurement. Animals were examined daily for clinical signs and mortality. Body weight and food consumption was measured weekly except mating period. After 14 days of treatment to both male and female, animals were paired (1:1) for maximum 14 days. The subsequent morning onwards, vaginal smears of females were checked to confirm the evidence of mating. After the confirmation of the mating, females were separated. Each litter was examined as soon as possible after delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted, sexed and litters weighed within 24 hours of parturition and on day 4 post-partum. Males and females were sacrificed on day 29 and post natal day 4, respectively and subjected to necropsy. Non pregnant females were sacrificed on their respective gestation day 26 after the evidence of mating or completion of mating period.

Clinical Signs and Mortality: Only sporadic clinical symptoms could be found during the treatment period. No mortality was recorded.

Body Weight Development: In males and females, there were no treatment related changes observed for the body weight and body weight change in treatment groups when compared to the control group.

Food Consumption: In males and females, there were no treatment related effects observed for food consumption during study period in treatment groups when compared to the control.

Litter Weight data: In males and females, there were no treatment related changes observed for the litter weight data in treatment groups when compared to the control group.

Precoital interval and duration of gestation: There was no treatment related effect observed for the duration of precoital interval and gestation days in treatment groups when compared to the control. All females in control and treatment groups showed evidence of copulation during 14 days mating period. The mating resulted in 100% pregnancy rate in C, MD, and HD groups as well as 90% in LD group. This finding was not considered to be test item related. 

Pre and post natal data: A tendency to an increase in the percentage of post implantation loss could be detected for LD, MD, and HD groups. However, no dose dependency as well as a high variability among the individual animals was detected. Hence, the toxicological relevance remains unclear.

Litter data

A tendency to a decreased sex ratio (m/f) was found in LD animals. However, this finding was not observed in MD and HD animals. Hence, it is assumed to be incidentally. 

Reproductive indices

There was no statistical significant difference observed for copulation index (%), fertility index (%), delivery index (%) and viability index (%) when compared to the control. Only one value (fertility index of LD animals) was below 100 % which results due to an individual animal which was not pregnant although a sperm positive smear was ascertained.

Pup survival data

One individual foetus of animal No. 75 (HD group) was found dead on PND 1. The viability index of this individual animal was 93.33 %. All other foetuses from this animal and the foetuses from all other animals survived till PND4. Hence, a viability index of 100% is given. Since only a single foetus from an individual animal was affected, this finding is assumed to be incidentally.

Pup External findings: Only two individual external findings were recorded. There was no dose response pattern observed for the type findings. Hence, they were considered incidental.

Gross Pathology: Only two individual macroscopic findings were recorded. There was no dose response pattern observed for the type findings. Hence, they were considered incidental.

Organ Weight: In both males and females, there was no treatment related changes observed for organ weight in treatment groups when compared to the corresponding control group.

Histopathology: No test item-related histological findings were noted in the male and in the female reproductive organs.

CONCLUSION:

The male Wistar rats after being treated with DMPPA_701-402-5 in aqua ad injectionem for the period of 28 days at the dosage of 100, 300 and 1000 mg/ kg body weight revealed no clear treatment related changes observed for the measured parameters when compared to the corresponding controls. Hence, the no observed adverse effect level (NOAEL) for male adult animals is assumed to be 1000 mg/kg body weight. The female rats were dose administered for approximately 54 days at the same dosage. There was no treatment related changes observed for any of the parameters when compared to the control. Hence, the no observed adverse effect level (NOAEL) for female adult animals is assumed to be 1000 mg/kg body weight. No treatment related changes could be evaluated for the foeti – neither for reproductive toxicology nor for developmental toxicology. Hence, the no observed adverse effect level (NOAEL) for reproductive toxicology as well as for developmental toxicology is believed to be 1000 mg/kg body weight.