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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 December 2001 - 24 May 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
22.03.1996
Deviations:
no
Principles of method if other than guideline:
The study was performed with two subgroups in parallel: the toxicity subgroup (results summarized in section 7.5, n=5) and the reproductive subgroup (results summarized in this section; n=5 for males, n=10 for females). The males in the toxicity subgroup were used as partners for the reproductive
subgroup females.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Batch No. ERC 20.12.2001/1
DAP: 17,93% total nitrogen; 46.86% available phosphoric acid expressed as % P2O5 equivalent (chemical analyses on October 19, 2001)
Storage: at room temperature

Test animals

Species:
rat
Strain:
other: CRL:CD(SD)IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 298-386 g (males), 191-263 g (females)
- Fasting period before study: No, except o/n before bloodsamples were taken
- Housing: single housing (except during mating) in RB3 modified cages, except for reproductive subgroup females from day 17 after mating,
where RB3 solid-bottomed cages were used
- Diet: expanded rodent diet, Rat and Mouse No. 1 Maintenance Diet (Special Diets Services Ltd, Witham, Essex, England), ad libitum (males); pelleted rodent diet, UAR VRF 1 certified (supplied by Charles River UK Limited, Margate, Kent, England) formulated as a breeding diet, ad libitum (females)
- Water: tap water, ad libitum
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS (target)
- Temperature (°C): 19-23
- Humidity (%): 40-70
- Air changes (per hr): appr. 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 28 December 2001 To: 24 May 2002

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The formulation for the high dose group was mixed by adding the required volume of water purified by reverse osmosis to the required weight of DAP and magnetically stirring for up to 1 hour until a visibly homogenous brown suspension was formed. Formulations of the other two dose groups were prepared by direct dilution of this suspension. Small amounts of dark meterial were apparent in the solution. This was considered to be due to the technical grade of the material, and formulations with small amounts of dark material present weere accepted for use in the study.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: up to 2 weeks (although all animals were mated and were seperated within 1 week)
- Proof of pregnancy: sperm in vaginal smear or at least three copulation plugs referred to as day 0 of pregnancy
- No unsuccessful pairing occurred
- Further matings after two unsuccessful attempts: no, not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of formulations prepared during weeks 1, 4 and 6 of the study were analysed for test material content.
Duration of treatment / exposure:
Males were treated until termination during week 6 of treatment. Doses were administered to the females for two weeks prior to pairing, throughout pairing and gestation until day 3 of lactation. Animals that were in parturition at the time of dosing were not dosed that day. Control animals received the vehicle over the same treatment period. Animals were not dosed on their scheduled day of necropsy.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Dose / conc.:
1 500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As no treatment-related effects were seen on parameters assessed in a preliminary study at 1000 mg/kg/day, the high dosage of 1500 mg/kg/day was selected to attempt to elicit an overt toxic response and establish a reference level for toxicity.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before start of the treatment and weekly during treatment
BODY WEIGHT: Yes
- Time schedule for examinations: start of treatment, weekly thereafter and at necropsy
- Reproductive subgroup females: on the first day of treatment, weekly until pairing and on Days 0, 7, 14, 17 and 20 after mating, and Days 1 and 4 of lactation, and at necropsy.
FOOD CONSUMPTION: Yes
- Food consumption for each animal determined per week (except during the time of mating for the males)
- Reproductive subgroup females: weekly before pairing then on Days 0,7,14, 17 and 20 after mating and Days 1 and 4 of lactation. No food consumption was recorded for toxicity subgroup males or reproductive subgroup animals during pairing.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 5, following functional observations
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All
- Parameters: according to OECD guideline
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
during week 5, following functional observations
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All
- Parameters: according to OECD guideline
FUNCTIONAL OBSERVATION BATTERY: Yes
- Time schedule for examinations: once after 4 weeks of treatment
- Dose groups that were examined: all
- Battery of functions tested: sensory activity, grip strength and motor activity

From day 20 after mating reproductive subgroup animals were checked 3 times daily for evidence of parturition. The females were permitted to deliver their young naturally and rear their own offspring until Day 4 of lactation. Numbers of live and dead offspring were recorded during the parturition process.
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
Testis weight, epididymis weight, histopathological examinations
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities and assessment of the stomach for milk content.
Postmortem examinations (parental animals):
Males were killed after the toxicity subgroup animals and the testes, epididymides, seminal vesicles with coagulating gland and prostate were retained for each animal. Females were killed on day 4 of lactation, the number of implantation sites was recorded and the ovaries, uterus with oviducts and cervix, vagina, pituitary and mammary tissue retained in appropriate fixative. Abnormal tissues were also retained in appropriate fixative.
Postmortem examinations (offspring):
SACRIFICE
Offspring of were killed by intraperitoneal injection of sodium pentobarbitone on day 4 of age and subjected to a macroscopic necropsy.
Any offspring dying before scheduled termination were subjected to necropsy and assessment of the stomach for milk.
GROSS NECROPSY
Gross necropsy consisted of detailed examination of the external features and orifices, the neck and associated tissues and the cranial, thoracic, abdominal and pelvic cavities and their viscera. Abnormal tissues were also retained in appropriate fixative.
HISTOPATHOLOGY / ORGAN WEIGTHS
Abnormalities observed at macroscopic necropsy.
Statistics:
See section 7.5.1
Reproductive indices:
Percentage mating = 100 * (animals with evidence of mating) / (animals paired)
Conception rate = 100 * (animals achieving a pregnancy) / (animals with evidence of mating)
Fertility index = 100 * (animals achieving a pregnancy) / (animals paired)
Gestation index = 100 * (number of live litters born) / (number pregnant)
Offspring viability indices:
Post-implantation survival index = 100 * (total number of offspring born) / (total number of implantation sites)
Live birth index = 100 * (total number of offspring on day 1 of littering) / (total number of offspring born)
Viability index = 100 * (total number of offspring on day 4 of littering) / (total number of offspring on day 1 of littering)
Sex ration = 100* (number of males in litter/total number of offspring in litter)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
A dose-dependent increase in transient post-dosing salivation was seen throughout the study, considered related to the palatability of dosing formulations administered via gavage. Dose-related increase in incidence of reddening of the extremities was seen, especially during the first week of treatment.
Mortality:
no mortality observed
Description (incidence):
No mortality occurred in the males and in the females in the reproduction group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Bodyweight gain for high dose males was decreased compared to the control males over the study period (- 22%), decreased weight gain was seen mainly in the fist week and last week of the study period. This effect is expected to be related to the irritant effects of the test item in the stomach, which is reflected in the decreased food intake of the high dose males throughout the study. Overall weight gain in females was the same for control and exposed rats.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption was slightly depressed for high dose males compared to control males (on average the high dose males consumed 6% less food each week). No effect on food consumption was seen in females.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food conversion efficiency was low for males at 1500 mg/kg bw/day throughout the study, but especially during week 5 compared with conrols and other groups (4.4%, 2.0%, 3.0% and 0.8% for controls and males exposed to 250, 750 and 1500 mg/kg bw/day, respectively). In females, no dose-related effect was seen on food conversion, however the percentages could not be calculated in week 5 for females at 750 and 1500 mg/kg bw/day due to the fact that female in these dose groups lost weight.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There was a reduction in activated partial thromboplastin time for toxicity subgroup males at 750 and 1500 mg/kg/day (74 and 76% of control, resp.), although this was not dosage related. No treatment related changes in these parameters were observed for toxicity subgroup females.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Alkaline phosphatase (ALP) levels were increased in all exposed males (compared to controls: +22%, +32.4% and +31% for the males exposed to 250, 750 and 1500 mg/kg bw/day, respectively). Glucose and total protein levels were depressed in high dose males (-21% (glucose) and -8.8% (protein) compared to controls), which is expected to be a reflection of the lower food intake observed. Also in females a dose-related increase was seen in ALP levels (compared to controls: +13% and +22% for the females exposed to 750 and 1500 mg/kg bw/day, respectively). Blood phosphorous levels were
decreased in high dose males (-18% compared to controls) and in exposed females (-10.9%, -12% and -19.1%).
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Motor activity was decreased in all exposed males compared to controls, no dose-relationship was observed. In females no difference was seen between exposed and controls, therefore this effect in males was not considerd to be related to the test item. Sensory reactivity and grip strength was comparable between control and exposed rats.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Substance-related effects were seen in the stomach of exposed males and females. Submucosal inflammation (glandular region was seen in 3/5, 4/5 and 2/5 males and 2/5, 4/5 and 4/5 females dosed at 250, 750 and 1500 mg/kg bwday, respectively. No effects weer seen in the stomach of control rats. Other effects seen in the stomach were submucosal oedema (glandular region) in one male and one female at 750 mg/kg bw/day and in two high dose females, increased mucus secreting cells in 2 males and 2 females at 250 mg/kg bw/day and 2 males and one female in the high dose group and acantholysis (non-glandular region), which was seen in one male at 750 and 1500 mg/kg bw/day each. Epithelial hyperplasia of the limiting ridge was seen in one male at 250 mg/kg bw/day, and two males at 750 and 1500 mg/kg bw/day each, and in 4 females at 750 mg/kg bw/day. These effects are considered to be related to the irritant effect of the test item formulations. Cortical tubular basophilia was noted in kidneys of two males of the high dose group. This was a lso seen in one control male. In one high dose male in addition cortical scarring, mineralisation of the cortex and hydronephrosis were noted in the kidney. As this was not seen in females, seen only in two males and severity was limited, this was not considered adverse. Focal inflammation with associated hepatocellular degeneration was seen in one male of the high dose group (and in one control female). Due to the low incience this observation was not considered to be related to the test item exposure.
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
All animals mated within 4 days (i.e. at fthe first oestrus opportunity after pairing), except one female in the high dose group that mated within 8 days. One control female did not get pregnant. All females gave birth to a live litter after 22-23 days of gestation. The number of uterine implantation sites (mean of 15.7, 15.7, 14.1 and 15.4 for controls, females exposed to 250, 750 and 1500 mg/kg bw/day, respectively), total litter size at day 1 (mean of 14.9, 14.7, 12.7 and 14.0 for controls, females exposed to 250, 750 and 1500 mg/kg bw/day, respectively) and live litter size at day 4 (mean of 14.6, 14.3, 12.7 and 14.0 for controls, females exposed to 250, 750 and 1500 mg/kg bw/day, respectively) were unaffected by the treatment.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects seen on reproduction and development at highest dose tested (1500 mg/kg bw/day).

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Description (incidence and severity):
Total litter size at day 1 and live litter size at days 1 and 4 were unaffected by the test item. The number of dead pups per nest was 2/2, 3/2, 0 and 0 for controls and groups exposed to 250, 750 and 1500 mg/kg bw/day, respectively.
Post-implantation surival indices of 95.2%, 93.5%, 90% and 94.8%, live birth indices of 99.3%, 99.4%, 100% and 95.9% and survival indices of 98.6%, 98.2%, 100% and 100% for controls, females exposed to 250, 750 and 1500 mg/kg bw/day, respectively.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Bodyweight increase from day 1 to day 4 was comparable for all groups (male offspring: mean body weight day 1: 6.4g, 6.3g, 6.6g, 6.3g, mean body weight change between days 1-4: 2.3g, 2.2g, 2.6g and 2.5g for controls, groups exposed to 250, 750 and 1500 mg/kg bw/day, respectively. Female offspring: mean body weight day 1: 5.9g, 6.0g, 6.1g, 6.0g, mean body weight change between days 1-4: 2.3g, 1.9g, 2.4g and 2.4g for controls, groups exposed to 250, 750 and 1500 mg/kg bw/day, respectively.
Other effects:
no effects observed
Description (incidence and severity):
The percentage of males in the litters did not indicate any preferential mortality amongst either sex (males day 1: 54.2%, 53%, 50% and 49.5% for controls, females exposed to 250, 750 and 1500 mg/kg bw/day, respectively) There were few macroscopic findings for offspring, these were minor in nature, and there were none that were considered to be related to treatment.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 500 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Sex:
male/female
Basis for effect level:
other: No effects seen on reproduction and development at highest dose tested (1500 mg/kg bw/day).

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

The mean concentrations of DAP in formulations, prepared for dosing during the first, fourth and last weeks of treatment, ranged from 98.0% to 103% of nominal concentrations.

Applicant's summary and conclusion

Conclusions:
A repeated dose toxicity study with screening for effects on reproduction and development was performed with diammonium phosphate in accordance with OECD 422 guidelines and GLP principles. Based on the results, the LOAEL of diammonium phosphate for local effects was concluded to be 250 mg/kg bw/day, the NOAEL for systemic toxicity was 750 mg/kg bw/day for subacute exposure. As no effects were seen on reproduction and development, the NOAEL for these parameters was established to exceed 1500 mg/kg bw/day.