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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

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. This value is read across to ammonium dihydrogen citrate, the NOAEL of the citrate part of ammonium dihydrogen citrate is expected to exceed the NOAEL for the ammonium part (see section 13 for the rationale). The corresponding NOAEL was calculated to be at least 7364 mg/kg bw/day for ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water).

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
The rationale to read across the data is attached in Section 13.
Reason / purpose for cross-reference:
read-across source
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: 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.
Dose descriptor:
NOAEL
Effect level:
>= 4 751 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
pure ammonium dihydrogen citrate
Sex:
male/female
Basis for effect level:
other: no effects seen on reproduction and development at highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 7 364 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water)
Sex:
male/female
Basis for effect level:
other: no effects seen on reproduction and development at highest dose tested.
Key result
Critical effects observed:
no
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.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 4 751 mg/kg bw/day (actual dose received)
Based on:
test mat. (total fraction)
Remarks:
ammonium dihydrogen citrate (recalculated value)
Sex:
male/female
Basis for effect level:
other: No effects seen on reproduction and development at highest dose tested (1500 mg/kg bw/day).
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

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.

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. This value is read across to ammonium dihydrogen citrate in water (see section 13 for the rationale). The corresponding NOAEL was calculated to be at least 7364 mg/kg bw/day for ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water).
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
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)
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
Species:
rat
Strain:
other: CRL:CD(SD)IGS BR
Sex:
male/female
Details on test animals or test system 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
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
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
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)
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: 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.
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).
Key result
Critical effects observed:
no
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.
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).
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

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.

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.
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
7 364 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is concluded to be reliable (Klimisch 1).
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
The study is considered reliable (Klimisch 1).
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

One study is available with substance analogue citric acid in which several species (rat, mouse, hamster and rabbit) were tested comparable to OECD 414. In none of the species maternal or developmental effects were seen up to and including the highest dose tested. The ammonium part of the registered substance was shown not to have adverse effects on development in a screening study. The data are read across to ammonium dihydrogen citrate in water (see Section 13).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
The rationale to read across the data is attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Clinical signs:
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control hamster and one high dose hamster died during the study. All other dams survived until sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Weight increase of exposed groups was comparable to negative control group (based on average value, standard deviations not included).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were observed.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The average number of implantation sites per dam was 13.8, 13.4, 13.3, 13.2 and 13.2 for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively. The average number of live fetuses per dam were 13.4, 13.0, 12.8, 12.7 and 12.9 for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively. The exposure to the test substance did not have effect on implantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
All pregnant dams had live litters.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The total number of resorptions was 8, 9, 5, 9 and 8 for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively. The number of dams with one or more sites resorbed was 5, 4, 4, 6 and 6 for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively.The percentage of dams with partial resorptions was 23.8%, 18.2%, 18.2%, 26.1% and 27.3% for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
No dead fetuses were found in the control group. In the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, 0, 7, 1 and 0 dead fetuses were found, respectively. The 7 dead fetuses were found in one nest. In absence of a dose-relationship, the dead fetuses were concidered not to be related to the test substance.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All pregnant dams that survived to term had live litters.
Dose descriptor:
NOAEL
Effect level:
>= 296.2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate (recalculated value)
Basis for effect level:
other: No effects seen up to and including the highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 459 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water)
Basis for effect level:
other: No effects seen up to and including the highest dose tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The average fetus weight was 1.78, 1.79, 1.81, 1.78 and 1.84 g for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio (M/F) for the live fetuses was 0.74, 0.81, 0.75, 0.77 and 0.67 for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external malformations were noted in any of the groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of skeletal malformations in the exposed groups did not differ from the control group (results summarized below).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 58.7 mg/kg bw/day, one pup was found with meningoencephalocele. No other visceral malformations were found in the groups exposed to citric acid and in the negative control group. This was regarded as an incidental finding.
Dose descriptor:
NOAEL
Effect level:
>= 296.2 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate (pure substance)
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 459 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water)
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Summary of skeletal findings (number of foetuses affected/ number of litters affected)

 

Control

2.72 mg/kg bw/day

12.6 mg/kg bw/day

58.7 mg/kg bw/day

272.0 mg/kg bw/day

Live fetuses examined (pregnant dams)

194 (21)

196 (22)

194 (22)

203 (23)

196 (22)

Sternebrae

Incomplete ossification

95/21

93/21

66/18

104/22

72/19

Bipartite

11/7

23/13

15/10

22/13

17/10

Extra

1/1

2/2

8/6

2/2

1/1

Missing

30/12

27/15

15/7

21/8

15/9

Ribs

Incomplete ossification

1/1

 

 

 

 

Fused/split

1/1

 

1/1

 

 

Wavy

3/3

 

 

 

 

Less than 12

2/1

 

 

 

 

More than 13

41/15

59/18

57/21

61/17

43/19

Vertebrae

Incomplete ossification

4/4

6/3

2/2

1/1

2/2

Skull

Incomplete closure

 

 

1/1

 

 

Extremities

Incomplete ossification

5/4

6/4

1/1

1/1

2/2

Miscellaneous

Hyoid, missing

4/4

3/2

1/1

 

1/1

Hyoid, reduced

4/3

4/3

6/4

4/4

2/2

 

Conclusions:
Hamsters were exposed to citric acid at 0, 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day via oral gavage for 5 consecutive days during pregnancy (gestation days 6 to 10). No effects were seen in maternal animals or fetuses. The nature and number of abnormalities seen in skeletal and soft tissues of the test groups did not differ from the controls. Based on these results, it is concluded that the NOAEL (maternal and development) is at least 272.0 mg/kg bw/day in hamsters. This result is read across to ammonium dihydrogen citrate in water. The corresponding NOAEL was calculated to be at least 459 mg/kg bw/day for ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water).
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
The rationale to read across the data is attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Clinical signs:
not specified
Mortality:
no mortality observed
Description (incidence):
All dams survived until sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Weight increase of exposed groups was comparable to negative control group (based on average value, standard deviations not included).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were observed.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The average number of implantation sites per dam was 11.9, 11.8, 11.8, 11.9 and 11.7 for the negative control group and the groups exposed to 2.41, 11.2, 52.0 and 241.0 mg/kg bw/day, respectively. The average number of live fetuses per dam were 11.2, 11.4, 11.1, 11.3 and 11.1 for the negative control group and the groups exposed to 2.41, 11.2, 52.0 and 241.0 mg/kg bw/day, respectively. The exposure to the test substance did not have effect on implantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
All pregnant dams had live litters.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The total number of resorptions was 10, 5, 14, 11 and 14 for the negative control group and the groups exposed to 2.41, 11.2, 52.0 and 241.0 mg/kg bw/day, respectively. The percentage of dams with partial resorptions was 45.0%, 21.7%, 54.6%, 36.4% and 41.7% for the negative control group and the groups exposed to 2.41, 11.2, 52.0 and 241.0 mg/kg bw/day, respectively.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Four dead fetuses were found in the control group. In the groups exposed to 2.41, 11.2 and 52.0 mg/kg bw/day, 3 dead fetuses ,1 and 1 dead fetus were found, respectively. The number of dams with one or more dead fetuses was 4 (control group) and 3 (2.41 mg/kg bw/day). No dead fetuses were found in the high dose group. In absence of a dose-relationship, the dead fetuses were concidered not to be related to the test substance.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All pregnant dams had live litters.
Dose descriptor:
NOAEL
Effect level:
>= 262.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate (recalculated value)
Basis for effect level:
other: No effects seen up to and including the highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 406.7 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
Ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water)
Basis for effect level:
other: No effects seen up to and including the highest dose tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The average fetus weight was 0.84, 0.83, 0.90, 0.94 and 0.87 g for the negative control group and the groups exposed to 2.41, 11.2, 52.0 and 241.0 mg/kg bw/day, respectively.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio (M/F) for the live fetuses was 1.01, 0.83, 0.90, 0.94 and 0.87 for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external malformations were noted in any of the groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of skeletal malformations in the exposed groups did not differ from the control group (results summarized below).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 52.0 mg/kg bw/day, one pup was found with meningoencephalocele. No other visceral malformations were found in the groups exposed to citric acid and in the negative control group. This was regarded as an incidental finding.
Dose descriptor:
NOAEL
Effect level:
>= 262.4 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 406.7 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water)
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Summary of skeletal findings (number of foetuses affected/ number of litters affected)

 

Control

2.41 mg/kg bw/day

11.2 mg/kg bw/day

52.0 mg/kg bw/day

241.0 mg/kg bw/day

Live fetuses examined (pregnant dams)

223 (20)

263 (23)

244 (22)

249 (22)

266 (24)

Sternebrae

Incomplete ossification

59/18

72/20

77/19

28/9

77/16

Bipartite

5/4

8/7

1/1

6/5

6/6

Extra

 

 

5/2

 

 

Missing

25/11

29/11

29/10

7/5

19/10

Ribs

Wavy

 

1/1

1/1

 

 

More than 13

35/12

46/16

38/16

43/15

49/16

Vertebrae

Incomplete ossification

8/3

9/6

7/4

1/1

5/1

Skull

Incomplete closure

9/3

6/4

5/2

 

4/1

Miscellaneous

Hyoid, missing

49/15

40/15

39/15

24/13

38/16

Hyoid, reduced

18/12

25/17

24/11

19/12

33/14
Conclusions:
Mice were exposed to citric acid at 0, 2.41, 11.2, 52.0 and 241.0 mg/kg bw/day via oral gavage for 10 consecutive days during pregnancy (gestation days 6 to 15). No effects were seen in maternal animals or fetuses. The nature and number of abnormalities seen in skeletal and soft tissues of the test groups did not differ from the controls. Based on these results, it is concluded that the NOAEL (maternal and development) is at least 241.0 mg/kg bw/day in mice. This result is read across to ammonium dihydrogen citrate in water. The corresponding NOAEL was calculated to be at least 406.7 mg/kg bw/day for ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water).
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
The rationale to read across the data is attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Clinical signs:
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One rabbit in the group exposed to 4.25 mg/kg bw/day died before day 29. This was considered an incidental finding.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Weight increase of exposed groups was comparable to negative control group (based on average value, standard deviations not included).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Two rabbits in the group exposed to 4.25 mg/kg bw/day aborted before day 29. As this occurred in one group only and not in the high dose group, this was considered an incidental finding.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The average number of Corpora Lutea per dam was 15.1, 13.2, 14.4, 11.1 and 14.4 for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively.The average number of implantation sites per dam was 5.44, 6.09, 6.30, 5.22 and 4.79 for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively. The average number of live fetuses per dam were 4.78, 4.91, 6.00, 5.22 and 4.36 for the negative control group and the groups exposed to4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
In the control group, and in the groups exposed to 4.25 and 425.0 mg/kg bw/day, there was one dam with all sites resorbed. This resulted in a percentage of dams with complete resorptions of 11.1%, 9.09% and 7.14% for the control group, and for the groups exposed to 4.25 and 425.0 mg/kg bw/day, respectively.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The total number of resorptions was 6, 13, 2, 0 and 4 for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively. The number of dams with one or more sites resorbed was 6, 4, 2, 0 and 3 for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively.The percentage of dams with partial resorptions was 66.7%, 36.4%, 20.0%, 0% and 21.4% for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One dead fetus was found in the group exposed to 19.75 mg/kg bw/day and 2 dead fetuses were found in the high dose group (from 2 nests). Due to the low number of dead fetuses, this was considered not to be related to substance exposure.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
All pregnant dams that survived to term had live litters, except for two dams at 4.25 mg/kg bw/day, which aborted before Day 29.
Dose descriptor:
NOAEL
Effect level:
>= 462.8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate (pure substance)
Basis for effect level:
other: No effects seen up to and including the highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 717 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water)
Basis for effect level:
other: No effects seen up to and including the highest dose tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The average fetus weight was 37.9, 36.6, 36.7, 37.6 and 35.0 g for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio (M/F) for the live fetuses was 0.95, 0.93, 1.07, 1.04 and 0.91 for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external malformations were noted in any of the groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of skeletal malformations in the exposed groups did not differ from the control group (results summarized below).
Visceral malformations:
no effects observed
Description (incidence and severity):
No visceral malformations were found in the groups exposed to citric acid and in the negative control group.
Dose descriptor:
NOAEL
Effect level:
>= 462.8 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate (pure substance, recalculated value)
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 717 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water)
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including the highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Summary of skeletal findings (number of foetuses affected/ number of litters affected)

 

Control

4.25 mg/kg bw/day

19.75 mg/kg bw/day

91.7 mg/kg bw/day

425.0 mg/kg bw/day

Live fetuses examined (pregnant dams)

43 (8)

54 (10)

51 (9)

47 (9*)

54 (12)

Sternebrae

Incomplete ossification

1/1

2/2

4/3

2/2

5/3

Bipartite

 

1/1

1/1

1/1

 

Fused

 

4/3

4/3

 

 

Extra

 

3/2

 

3/3

2/2

Missing

 

 

7/2

3/1

 

 

* One litter lost in processing

Conclusions:
Rabbits were exposed to citric acid at 0, 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day via oral gavage for 13 consecutive days (day 6 to day 18 of gestation). Rabbits were sacrificed on Day 29 of gestation. No effects were seen in maternal animals or fetuses. The nature and number of abnormalities seen in skeletal tissues of the test groups did not differ from the controls (no soft tisue malformations were seen). Based on these results, it is concluded that the NOAEL (maternal and development) is at least 425.0 mg/kg bw/day in rabbits. This result is read across to ammonium dihydrogen citrate in water. The corresponding NOAEL was calculated to be at least 717 mg/kg bw/day for ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water).
Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
The rationale to read across the data is attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Vehicle:
water
Clinical signs:
not specified
Mortality:
no mortality observed
Description (incidence):
All dams survived until sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Weight increase of exposed groups was comparable to negative control group (based on average value, standard deviations not included).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were observed.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The average number of implantation sites per dam was 10.5, 11.8, 12.0, 12.3 and 11.7 for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively. The average number of live fetuses per dam were 10.2, 11.8, 11.9, 12.0 and 11.6 for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively. The exposure to the test substance did not have effect on implantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
All pregnant dams had live litters.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The total number of resorptions was 5, 0, 1, 5 and 3 for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively. The percentage of dams with partial resorptions was 14.3%, 0%, 4.35%, 5.26% and 14.3% for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One dead fetus was found in the group exposed to 63.6 mg/kg bw/day. This is considered an incidental finding.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All pregnant dams had live litters.
Dose descriptor:
NOAEL
Effect level:
>= 321.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate (pure substance)
Basis for effect level:
other: No effects seen up to and including the highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 406.7 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water)
Basis for effect level:
other: No effects seen up to and including the highest dose tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The average fetus weight was 3.87, 3.72, 3.99, 3.96 and 3.99 g for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio (M/F) for the live fetuses was 0.92, 1.10, 1.06, 1.10 and 0,85 for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of external malformations in the exposed groups did not differ from the control group (results summarized below).
Skeletal malformations:
no effects observed
Description (incidence and severity):
No external malformations were noted in any of the groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No visceral malformations were found in the groups exposed to citric acid and in the negative control group.
Dose descriptor:
NOAEL
Effect level:
>= 321.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate (recalculated value)
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 498 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water)
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Summary of skeletal findings (number of foetuses affected/ number of litters affected)

 

Control

2.95 mg/kg bw/day

13.7 mg/kg bw/day

63.6 mg/kg bw/day

295.0 mg/kg bw/day

Live fetuses examined (pregnant dams)

153 (21)

197 (24)

191 (23)

161 (19)

167 (21)

Sternebrae

 

 

 

 

 

Incomplete ossification

44/12

90/22

87/20

56/16

61/17

Bipartite

1/1

1/1

1/1

1/1

 

Missing

9/5

4/3

8/2

6/3

7/5

Ribs

 

 

 

 

 

Incomplete ossification

44/12

90/22

87/20

56/16

61/17

Wavy

19/9

19/10

18/11

16/10

17/8

More than 13

 

1/1

 

1/1

4/2

Vertebrae

 

 

 

 

 

Incomplete ossification

27/9

8/8

7/4

5/5

7/5

Skull

 

 

 

 

 

Incomplete closure

27/9

23/14

33/13

30/12

56/17

Extremities

Missing

 

4/1

 

 

 

Miscellaneous

Hyoid, missing

20/7

13/7

17/8

22/11

12/8

Hyoid, reduced

20/11

28/10

35/17

10/6

15/10

 

Conclusions:
Rats were exposed to citric acid at 0, 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day via oral gavage for 10 consecutive days during pregnancy (gestation days 6 to 15). No effects were seen in maternal animals or fetuses. The nature and number of abnormalities seen in skeletal tissues of the test groups did not differ from the controls. No visceral abnormalities were seen in control and exposed rats. Based on these results, it is concluded that the NOAEL (maternal and development) is at least 295.0 mg/kg bw/day in rats. This result is read across to ammonium dihydrogen citrate in water. The corresponding NOAEL was calculated to be at least 498 mg/kg bw/day for ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water).
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1973
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline"
Principles of method if other than guideline:
Exposure started gestation day 6 (recommended start on day 5), dose level choice not justified (highest dose tested did not induce maternal effects), number of corpora lutea not determined, limited information on test substance included in report. No information on feed and environmental conditions, no analytical verification of doses, only average fetus weight males and females together per litter, no individual information on the pups, no historical control data, limited maternal toxicity parameters.
GLP compliance:
no
Remarks:
Study performed before GLP principles were implemented.
Limit test:
no
Species:
hamster
Strain:
other: Golden hamster
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Housing: Individually housed in mesh bottom cages
- Diet: ad libitum
- Water: fresh tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- The cages were kept in temperature and humidity-controlled quarters
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Hamsters were dosed 2 mL/kg bw (high dose) or at 1 mL/kg bw.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Females were mated with young adult males (1 to 1), and appearance of motile sperm in the vaginal smear was considered Day 0 of gestation.
Duration of treatment / exposure:
Five consecutive days (day 6 to day 10 of gestation).
Frequency of treatment:
Daily gavage.
Duration of test:
On gestation Day 14, all dams were sacrificed.
Dose / conc.:
2.72 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
Dose / conc.:
12.6 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
Dose / conc.:
58.7 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
Dose / conc.:
272 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
No. of animals per sex per dose:
Pregnant females: 22 (negative control), 19-24 (positive control groups), 22 (groups exposed to 2.72 and 12.6 mg/kg bw/day), 23 (groups exposed to 58.7 and 272.0 mg/kg bw/day).
Control animals:
yes
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Appearance and behaviour with particular attention to food consumption and weight.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 8, 10, and 14

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 14
- Examinations: Number of implantation sites, resorption sites, live and dead fetuses; The urogenital tract of each dam was examined in detail for anatomical abnormalities.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
Number of implantation sites, resorption sites, live and dead fetuses
Fetal examinations:
- Body weight (live pups)
- External examinations: Yes: All per litter
- Soft tissue examinations: Yes: One-third per litter
- Skeletal examinations: Yes: Two-third per litter
Clinical signs:
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control hamster and one high dose hamster died during the study. All other dams survived until sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Weight increase of exposed groups was comparable to negative control group (based on average value, standard deviations not included).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were observed.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The average number of implantation sites per dam was 13.8, 13.4, 13.3, 13.2 and 13.2 for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively. The average number of live fetuses per dam were 13.4, 13.0, 12.8, 12.7 and 12.9 for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively. The exposure to the test substance did not have effect on implantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
All pregnant dams had live litters.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The total number of resorptions was 8, 9, 5, 9 and 8 for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively. The number of dams with one or more sites resorbed was 5, 4, 4, 6 and 6 for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively.The percentage of dams with partial resorptions was 23.8%, 18.2%, 18.2%, 26.1% and 27.3% for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
No dead fetuses were found in the control group. In the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, 0, 7, 1 and 0 dead fetuses were found, respectively. The 7 dead fetuses were found in one nest. In absence of a dose-relationship, the dead fetuses were concidered not to be related to the test substance.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All pregnant dams that survived to term had live litters.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 272 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No effects seen up to and including the highest dose tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The average fetus weight was 1.78, 1.79, 1.81, 1.78 and 1.84 g for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio (M/F) for the live fetuses was 0.74, 0.81, 0.75, 0.77 and 0.67 for the negative control group and the groups exposed to 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day, respectively.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external malformations were noted in any of the groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of skeletal malformations in the exposed groups did not differ from the control group (results summarized below).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 58.7 mg/kg bw/day, one pup was found with meningoencephalocele. No other visceral malformations were found in the groups exposed to citric acid and in the negative control group. This was regarded as an incidental finding.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 272 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Summary of skeletal findings (number of foetuses affected/ number of litters affected)

 

Control

2.72 mg/kg bw/day

12.6 mg/kg bw/day

58.7 mg/kg bw/day

272.0 mg/kg bw/day

Live fetuses examined (pregnant dams)

194 (21)

196 (22)

194 (22)

203 (23)

196 (22)

Sternebrae

Incomplete ossification

95/21

93/21

66/18

104/22

72/19

Bipartite

11/7

23/13

15/10

22/13

17/10

Extra

1/1

2/2

8/6

2/2

1/1

Missing

30/12

27/15

15/7

21/8

15/9

Ribs

Incomplete ossification

1/1

 

 

 

 

Fused/split

1/1

 

1/1

 

 

Wavy

3/3

 

 

 

 

Less than 12

2/1

 

 

 

 

More than 13

41/15

59/18

57/21

61/17

43/19

Vertebrae

Incomplete ossification

4/4

6/3

2/2

1/1

2/2

Skull

Incomplete closure

 

 

1/1

 

 

Extremities

Incomplete ossification

5/4

6/4

1/1

1/1

2/2

Miscellaneous

Hyoid, missing

4/4

3/2

1/1

 

1/1

Hyoid, reduced

4/3

4/3

6/4

4/4

2/2

 

Conclusions:
Hamsters were exposed to citric acid at 0, 2.72, 12.6, 58.7 and 272.0 mg/kg bw/day via oral gavage for 5 consecutive days during pregnancy (gestation days 6 to 10). No effects were seen in maternal animals or fetuses. The nature and number of abnormalities seen in skeletal and soft tissues of the test groups did not differ from the controls. Based on these results, it is concluded that the NOAEL (maternal and development) is at least 272.0 mg/kg bw/day in hamsters.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1973
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline"
Principles of method if other than guideline:
Exposure started gestation day 6 (recommended start on day 5), dose level choice not justified (highest dose tested did not induce maternal effects), number of corpora lutea not determined, limited information on test substance included in report. No information on feed and environmental conditions, no analytical verification of doses, only average fetus weight males and females together per litter, no individual information on the pups, no historical control data, limited maternal toxicity parameters.
GLP compliance:
no
Remarks:
Study performed before GLP principles were implemented.
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Housing: Individually housed in mesh bottom cages
- Diet: ad libitum
- Water: fresh tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- The cages were kept in temperature and humidity-controlled quarters
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Rats were dosed at 10 mL/kg bw.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Females were mated with young adult males, and observation of the vaginal sperm plug was considered Day 0 of gestation.
Duration of treatment / exposure:
Ten consecutive days (day 6 to day 15 of gestation).
Frequency of treatment:
Daily gavage.
Duration of test:
On gestation Day 17, all dams were sacrificed.
Dose / conc.:
2.41 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
Dose / conc.:
11.2 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
Dose / conc.:
52 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
Dose / conc.:
241 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
No. of animals per sex per dose:
Pregnant females: 20 (negative control), 18 (positive control), 23 (group exposed to 2.41 mg/kg bw/day), 22 (groups exposed to 11.2 and 52.0 mg/kg bw/day) and 24 (group exposed to 241.0 mg/kg bw/day).
Control animals:
yes
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Appearance and behaviour with particular attention to food consumption and weight.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 6, 11, 15 and 17

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 17
- Examinations: Number of implantation sites, resorption sites, live and dead fetuses; The urogenital tract of each dam was examined in detail for anatomical abnormalities.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
Number of implantation sites, resorption sites, live and dead fetuses
Fetal examinations:
- Body weight (live pups)
- External examinations: Yes: All per litter
- Soft tissue examinations: Yes: One-third per litter
- Skeletal examinations: Yes: Two-third per litter
Clinical signs:
not specified
Mortality:
no mortality observed
Description (incidence):
All dams survived until sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Weight increase of exposed groups was comparable to negative control group (based on average value, standard deviations not included).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were observed.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The average number of implantation sites per dam was 11.9, 11.8, 11.8, 11.9 and 11.7 for the negative control group and the groups exposed to 2.41, 11.2, 52.0 and 241.0 mg/kg bw/day, respectively. The average number of live fetuses per dam were 11.2, 11.4, 11.1, 11.3 and 11.1 for the negative control group and the groups exposed to 2.41, 11.2, 52.0 and 241.0 mg/kg bw/day, respectively. The exposure to the test substance did not have effect on implantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
All pregnant dams had live litters.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The total number of resorptions was 10, 5, 14, 11 and 14 for the negative control group and the groups exposed to 2.41, 11.2, 52.0 and 241.0 mg/kg bw/day, respectively. The percentage of dams with partial resorptions was 45.0%, 21.7%, 54.6%, 36.4% and 41.7% for the negative control group and the groups exposed to 2.41, 11.2, 52.0 and 241.0 mg/kg bw/day, respectively.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
Four dead fetuses were found in the control group. In the groups exposed to 2.41, 11.2 and 52.0 mg/kg bw/day, 3 dead fetuses ,1 and 1 dead fetus were found, respectively. The number of dams with one or more dead fetuses was 4 (control group) and 3 (2.41 mg/kg bw/day). No dead fetuses were found in the high dose group. In absence of a dose-relationship, the dead fetuses were concidered not to be related to the test substance.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All pregnant dams had live litters.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 241 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No effects seen up to and including the highest dose tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The average fetus weight was 0.84, 0.83, 0.90, 0.94 and 0.87 g for the negative control group and the groups exposed to 2.41, 11.2, 52.0 and 241.0 mg/kg bw/day, respectively.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio (M/F) for the live fetuses was 1.01, 0.83, 0.90, 0.94 and 0.87 for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external malformations were noted in any of the groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of skeletal malformations in the exposed groups did not differ from the control group (results summarized below).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 52.0 mg/kg bw/day, one pup was found with meningoencephalocele. No other visceral malformations were found in the groups exposed to citric acid and in the negative control group. This was regarded as an incidental finding.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 241 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Summary of skeletal findings (number of foetuses affected/ number of litters affected)

 

Control

2.41 mg/kg bw/day

11.2 mg/kg bw/day

52.0 mg/kg bw/day

241.0 mg/kg bw/day

Live fetuses examined (pregnant dams)

223 (20)

263 (23)

244 (22)

249 (22)

266 (24)

Sternebrae

Incomplete ossification

59/18

72/20

77/19

28/9

77/16

Bipartite

5/4

8/7

1/1

6/5

6/6

Extra

 

 

5/2

 

 

Missing

25/11

29/11

29/10

7/5

19/10

Ribs

Wavy

 

1/1

1/1

 

 

More than 13

35/12

46/16

38/16

43/15

49/16

Vertebrae

Incomplete ossification

8/3

9/6

7/4

1/1

5/1

Skull

Incomplete closure

9/3

6/4

5/2

 

4/1

Miscellaneous

Hyoid, missing

49/15

40/15

39/15

24/13

38/16

Hyoid, reduced

18/12

25/17

24/11

19/12

33/14
Conclusions:
Mice were exposed to citric acid at 0, 2.41, 11.2, 52.0 and 241.0 mg/kg bw/day via oral gavage for 10 consecutive days during pregnancy (gestation days 6 to 15). No effects were seen in maternal animals or fetuses. The nature and number of abnormalities seen in skeletal and soft tissues of the test groups did not differ from the controls. Based on these results, it is concluded that the NOAEL (maternal and development) is at least 241.0 mg/kg bw/day in mice.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1973
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline"
Principles of method if other than guideline:
Dose level choice not justified (highest dose tested did not induce maternal effects), limited information on test substance included in report. No information on feed and environmental conditions, no analytical verification of doses, only average fetus weight males and females together per litter, no individual information on the pups, no historical control data, limited maternal toxicity parameters.
GLP compliance:
no
Remarks:
Study performed before GLP principles were implemented.
Limit test:
no
Species:
rabbit
Strain:
other: Dutch-belted rabbit
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Housing: Individually housed in mesh bottom cages
- Diet: ad libitum
- Water: fresh tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- The cages were kept in temperature and humidity-controlled quarters
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Rabbits were dosed 2 mL/kg bw (high dose) or at 1 mL/kg bw.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
On Day 0, each doe was given an injection of 0.4 ml of human chorionic gonadotropin (400 IU) via the marginal ear vein. Three hours later, each
doe was inseminated artificially with 0.3 ml of diluted semen from a proven donor buck using approximately 20 x 10E6 motile sperm according
to the procedure described by vogin et al (Pharmacologist 11, 282 (1969)).
Duration of treatment / exposure:
Thirteen consecutive days (day 6 to day 18 of gestation).
Frequency of treatment:
Daily gavage.
Duration of test:
On gestation Day 29, all dams were sacrificed.
Dose / conc.:
4.25 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
Dose / conc.:
19.75 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
Dose / conc.:
91.9 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
Dose / conc.:
425 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
No. of animals per sex per dose:
Pregnant females: 9 (negative control), 10 (positive control group), 14 (groups exposed to 4.25 and 425.0 mg/kg bw/day), 10 (group exposed to 91.7) a and 9 (group exposed to 425.0 mg/kg bw/day).
Control animals:
yes
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Appearance and behaviour with particular attention to food consumption and weight.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 6, 12, 18, and 29

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 29
- Examinations: Number of implantation sites, resorption sites, live and dead fetuses; The urogenital tract of each dam was examined in detail for anatomical abnormalities.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
Number of implantation sites, resorption sites, live and dead fetuses
Fetal examinations:
- Body weight (live pups)
- External examinations: Yes: All per litter
- Soft tissue examinations: Yes: One-third per litter
- Skeletal examinations: Yes: Two-third per litter
- After Caesarean section, the live fetuses of each litter were then placed in an incubator for 24 hours for the evaluation of neonatal survival.
Clinical signs:
not specified
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One rabbit in the group exposed to 4.25 mg/kg bw/day died before day 29. This was considered an incidental finding.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Weight increase of exposed groups was comparable to negative control group (based on average value, standard deviations not included).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
Two rabbits in the group exposed to 4.25 mg/kg bw/day aborted before day 29. As this occurred in one group only and not in the high dose group, this was considered an incidental finding.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The average number of Corpora Lutea per dam was 15.1, 13.2, 14.4, 11.1 and 14.4 for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively.The average number of implantation sites per dam was 5.44, 6.09, 6.30, 5.22 and 4.79 for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively. The average number of live fetuses per dam were 4.78, 4.91, 6.00, 5.22 and 4.36 for the negative control group and the groups exposed to4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
In the control group, and in the groups exposed to 4.25 and 425.0 mg/kg bw/day, there was one dam with all sites resorbed. This resulted in a percentage of dams with complete resorptions of 11.1%, 9.09% and 7.14% for the control group, and for the groups exposed to 4.25 and 425.0 mg/kg bw/day, respectively.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The total number of resorptions was 6, 13, 2, 0 and 4 for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively. The number of dams with one or more sites resorbed was 6, 4, 2, 0 and 3 for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively.The percentage of dams with partial resorptions was 66.7%, 36.4%, 20.0%, 0% and 21.4% for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One dead fetus was found in the group exposed to 19.75 mg/kg bw/day and 2 dead fetuses were found in the high dose group (from 2 nests). Due to the low number of dead fetuses, this was considered not to be related to substance exposure.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
All pregnant dams that survived to term had live litters, except for two dams at 4.25 mg/kg bw/day, which aborted before Day 29.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 425 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No effects seen up to and including the highest dose tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The average fetus weight was 37.9, 36.6, 36.7, 37.6 and 35.0 g for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio (M/F) for the live fetuses was 0.95, 0.93, 1.07, 1.04 and 0.91 for the negative control group and the groups exposed to 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day, respectively.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No external malformations were noted in any of the groups.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of skeletal malformations in the exposed groups did not differ from the control group (results summarized below).
Visceral malformations:
no effects observed
Description (incidence and severity):
No visceral malformations were found in the groups exposed to citric acid and in the negative control group.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 425 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Summary of skeletal findings (number of foetuses affected/ number of litters affected)

 

Control

4.25 mg/kg bw/day

19.75 mg/kg bw/day

91.7 mg/kg bw/day

425.0 mg/kg bw/day

Live fetuses examined (pregnant dams)

43 (8)

54 (10)

51 (9)

47 (9*)

54 (12)

Sternebrae

Incomplete ossification

1/1

2/2

4/3

2/2

5/3

Bipartite

 

1/1

1/1

1/1

 

Fused

 

4/3

4/3

 

 

Extra

 

3/2

 

3/3

2/2

Missing

 

 

7/2

3/1

 

 

* One litter lost in processing

Conclusions:
Rabbits were exposed to citric acid at 0, 4.25, 19.75, 91.7 and 425.0 mg/kg bw/day via oral gavage for 13 consecutive days (day 6 to day 18 of gestation). Rabbits were sacrificed on Day 29 of gestation. No effects were seen in maternal animals or fetuses. The nature and number of abnormalities seen in skeletal tissues of the test groups did not differ from the controls (no soft tisue malformations were seen). Based on these results, it is concluded that the NOAEL (maternal and development) is at least 425.0 mg/kg bw/day in rabbits.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1973
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline"
Principles of method if other than guideline:
Exposure started gestation day 6 (recommended start on day 5), dose level choice not justified (highest dose tested did not induce maternal effects), number of corpora lutea not determined, limited information on test substance included in report. No information on feed and environmental conditions, no analytical verification of doses, only average fetus weight males and females together per litter, no individual information on the pups, no historical control data, limited maternal toxicity parameters.
GLP compliance:
no
Remarks:
Study performed before GLP principles were implemented.
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Housing: gang-housed in disposable plastic cages
- Diet: ad libitum
- Water: fresh tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- The cages were kept in temperature and humidity-controlled quarters
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Rats were dosed at 1 mL/kg bw or 2 mL/kg bw (highest dose group only).
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Females were mated with young adult males, and observation of the vaginal sperm plug was considered Day 0 of gestation.
Duration of treatment / exposure:
Ten consecutive days (day 6 to day 15 of gestation).
Frequency of treatment:
Daily gavage.
Duration of test:
On gestation Day 20, all dams were sacrificed.
Dose / conc.:
2.95 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
Dose / conc.:
13.7 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
Dose / conc.:
63.6 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
Dose / conc.:
295 mg/kg bw/day (actual dose received)
Remarks:
No analytical verification of exposure.
No. of animals per sex per dose:
Pregnant females: 21 (negative control and group exposed to 295.0 mg/kg bw/day), 24 (positive control and group exposed to 2.95 mg/kg bw/day), 23 (group exposed to 13.7 mg/kg bw/day), 19 (group exposed to 63.6 mg/kg bw/day)
Control animals:
yes
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Appearance and behaviour with particular attention to food consumption and weight.

BODY WEIGHT: Yes
- Time schedule for examinations: Gestation days 0, 6, 11, 15 and 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20
- Examinations: Number of implantation sites, resorption sites, live and dead fetuses; The urogenital tract of each dam was examined in detail for anatomical abnormalities.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
Number of implantation sites, resorption sites, live and dead fetuses
Fetal examinations:
- Body weight (live pups)
- External examinations: Yes: All per litter
- Soft tissue examinations: Yes: One-third per litter
- Skeletal examinations: Yes: Two-third per litter
Clinical signs:
not specified
Mortality:
no mortality observed
Description (incidence):
All dams survived until sacrifice.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Weight increase of exposed groups was comparable to negative control group (based on average value, standard deviations not included).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not specified
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
No abortions were observed.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The average number of implantation sites per dam was 10.5, 11.8, 12.0, 12.3 and 11.7 for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively. The average number of live fetuses per dam were 10.2, 11.8, 11.9, 12.0 and 11.6 for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively. The exposure to the test substance did not have effect on implantation loss.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
All pregnant dams had live litters.
Early or late resorptions:
no effects observed
Description (incidence and severity):
The total number of resorptions was 5, 0, 1, 5 and 3 for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively. The percentage of dams with partial resorptions was 14.3%, 0%, 4.35%, 5.26% and 14.3% for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively.
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
One dead fetus was found in the group exposed to 63.6 mg/kg bw/day. This is considered an incidental finding.
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All pregnant dams had live litters.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 295 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No effects seen up to and including the highest dose tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The average fetus weight was 3.87, 3.72, 3.99, 3.96 and 3.99 g for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively.
Reduction in number of live offspring:
not examined
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex ratio (M/F) for the live fetuses was 0.92, 1.10, 1.06, 1.10 and 0,85 for the negative control group and the groups exposed to 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day, respectively.
Changes in litter size and weights:
not examined
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The number of external malformations in the exposed groups did not differ from the control group (results summarized below).
Skeletal malformations:
no effects observed
Description (incidence and severity):
No external malformations were noted in any of the groups.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No visceral malformations were found in the groups exposed to citric acid and in the negative control group.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 295 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including highest dose tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Summary of skeletal findings (number of foetuses affected/ number of litters affected)

 

Control

2.95 mg/kg bw/day

13.7 mg/kg bw/day

63.6 mg/kg bw/day

295.0 mg/kg bw/day

Live fetuses examined (pregnant dams)

153 (21)

197 (24)

191 (23)

161 (19)

167 (21)

Sternebrae

 

 

 

 

 

Incomplete ossification

44/12

90/22

87/20

56/16

61/17

Bipartite

1/1

1/1

1/1

1/1

 

Missing

9/5

4/3

8/2

6/3

7/5

Ribs

 

 

 

 

 

Incomplete ossification

44/12

90/22

87/20

56/16

61/17

Wavy

19/9

19/10

18/11

16/10

17/8

More than 13

 

1/1

 

1/1

4/2

Vertebrae

 

 

 

 

 

Incomplete ossification

27/9

8/8

7/4

5/5

7/5

Skull

 

 

 

 

 

Incomplete closure

27/9

23/14

33/13

30/12

56/17

Extremities

Missing

 

4/1

 

 

 

Miscellaneous

Hyoid, missing

20/7

13/7

17/8

22/11

12/8

Hyoid, reduced

20/11

28/10

35/17

10/6

15/10

 

Conclusions:
Rats were exposed to citric acid at 0, 2.95, 13.7, 63.6 and 295.0 mg/kg bw/day via oral gavage for 10 consecutive days during pregnancy (gestation days 6 to 15). No effects were seen in maternal animals or fetuses. The nature and number of abnormalities seen in skeletal tissues of the test groups did not differ from the controls. No visceral abnormalities were seen in control and exposed rats.
Based on these results, it is concluded that the NOAEL (maternal and development) is at least 295.0 mg/kg bw/day in rats.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
406.7 mg/kg bw/day
Study duration:
subacute
Species:
mouse
Quality of whole database:
The available study is performed comparable to OECD 414, but with some deficiencies. Since positive controls were included and several concentrations and species were tested, the study is evaluated as Klimisch 2.
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

Mice were exposed to citric acid at 0, 2.41, 11.2, 52.0 and 241.0 mg/kg bw/day via oral gavage for 10 consecutive days during pregnancy (gestation days 6 to 15). No effects were seen in maternal animals or fetuses. The nature and number of abnormalities seen in skeletal and soft tissues of the test groups did not differ from the controls. Based on these results, it is concluded that the NOAEL (maternal and development) is at least 241.0 mg/kg bw/day in mice. This result is read across to ammnoium dihydrogen citrate. The corresponding NOAEL was calculated to be at least 406.7 mg/kg bw/day for ammonium dihydrogen citrate in water (≤ 55% ammonium dihydrogen citrate and ≥ 45% water).

Parallel to mice, the experiment was also performed in rats, hamsters and rabbits. Rats (n= 19-24) were exposed for 10 consecutive days at a maximum dose of 295.0 mg/kg bw/day (gestation day 6 -15). Hamsters (n= 21-23) were exposed for 5 consecutive days at a maximum dose of 272 mg/kg bw/day (gestation day 6 – 10).Rabbits (n= 9-14) were exposed for 13 consecutive days at a maximum dose of 425 mg/kg bw/day (gestation day 6 – 18). For all test animals appropriate positive controls were included that showed that the experimental set-up was sufficient to detect induction of test substance-related abnormalities during fetal development. None of the citric acid exposed groups showed maternal toxicity, or effects on development different from the negative control group.

Justification for classification or non-classification

Based on the current data-set, Ammonium dihydrogen citrate in water is not classified for adverse effects on reproduction and development according to Regulation (EC) No. 1272/2008 and its amendments.

Additional information