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Developmental toxicity / teratogenicity

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Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
12 December 2001 - 24 May 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In accordance with REACH Annex XI, Section 1.5, of Regulation (EC) No. 1907/2006 (REACH) the standard testing regime may be adapted in cases where a grouping or read-across approach has been applied.

The similarities may be based on:
(1) a common functional group
(2) the common precursors and/or the likelihood of common breakdown products via physical or biological processes, which result in structurally similar chemicals; or
(3) a constant pattern in the changing of the potency of the properties across the category

The proposed source chemical (diammonium hydrogenorthophosphate) is highly soluble in water (> 10000 mg/L). In aqueous media soluble inorganic orthophosphates will dissociate to their ionic constituents; in this case ammonium and orthophosphate ions. Diammonium dihydrogenpyrophosphate will dissociate to ammonium cations and pyrophosphate anions. The pyrophosphate anions are unstable in aqueous solutions with the degree of instability varying according to pH. In distilled water they will hydrolyse slowly via abiotic mechanisms to orthophosphate. In natural waters a number of different processes can occur; abiotic hydrolysis, biotic degradation (as a result of the action of phosphatases which cleave pyrophosphates into orthophosphate subunits) and assimilation by organisms in the water. Thus, the target substance (diammonium dihydrogenpyrophosphate) and the source substance (diammonium hydrogenorthophosphate) will be primarily absorbed as the same inorganic ions: ammonium and orthophosphate and are expected to behave in a similar manner under test conditions.
All (bio) transformation products of the source chemical are common to the target chemical and as such the data is considered to be adequate and reliable for use in the assessment of diammonium dihydrogenpyrophosphate for the toxicity hazard assessment.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See read-across justification report attached.

3. ANALOGUE APPROACH JUSTIFICATION
See read-across justification report attached.

4. DATA MATRIX
See read-across justification report attached.
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD 422
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Diammonium hydrogenorthophosphate
EC Number:
231-987-8
EC Name:
Diammonium hydrogenorthophosphate
Cas Number:
7783-28-0
Molecular formula:
H3N.1/2H3O4P
IUPAC Name:
diammonium hydrogen phosphate
Details on test material:
- Name of test material (as cited in study report): Diammonium phosphate (DAP)
- Substance type: solid
- Physical state: solid
- Analytical purity: unknown
- Lot/batch No.: unknown
- Expiration date of the lot/batch: unknown
- Stability under test conditions: no data
- Storage condition of test material: Room temperature

- Other: The test substance was assigned ABC reference number TS-12397 upon arrival. The range-finding test was conducted with TS-12397.
Prior to the definitive test, the Sponsor Representative instructed ABC Laboratories, Inc., to process a subsample of the test substance into
a powder by use of a mortar and pestle. The processed subsample was assigned ABC reference number TS-12489.
The definitive test was conducted with TS-12489.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England.
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 298 to 386 g for males and 191 to 263 g for females
- Fasting period before study: not applicable
- Housing:Unless paired for mating, the animals were singly housed in RB3 modified cages consisiting of high density polypropylene bodies with lids and floors of stainless steel grid. These cages were suspended in batteries over trays lined with absorbent paper. RB3 modified cages were used
throughout the study with the exception of reproductive subgroup females from Day 17 after mating, where RB3 solid-bottomed cages were used.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C, except on 3 occasions during which the temperature was minimally 16 or 18°C and the target temperature range was
re-established within 24 hours.
- Humidity (%): 40-70%, except on 1 occasion during which the humidity was minimally 16% and the target temperature range was re-established on
the next day.
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 12 December 2001 To: 10 February 2002

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The formulation for Group 4 (150 mg/ml) was mixed by adding the required volume of vehicle (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. Formulation for Groups 3 and 2
were prepared by direct dilution of this suspension.

VEHICLE
- Concentration in vehicle: 25 - 75 - 150 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration during Weeks 1, 4 and 6 of the study were analysed using spectrophotometry for test
material content and found to be satisfactory.
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.
- After successful mating each pregnant female was caged (how): singly housed in RB3 solid-bottomed cages.
- Any other deviations from standard protocol: no.
Duration of treatment / exposure:
Animals were divided between two subgroups (toxicity and reproductive subgroups).
Males of both subgroups and females of the toxicity subgroup were treated until termination during week 6 of treatment. Doses were administered to the reproductive subgroup 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
No. of animals per sex per dose:
The toxicity subgroup consisted of 5 males and 5 females per group and the reproductive subgroup consisted of 10 females and
5 males per group.
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 by the Sponsor to attempt to elicit an overt toxic response and establish a reference level for toxicity.
The concept of a 'Limit Dose' is accepted by the OECD and is generally set in the region of 1000 mg/kg/day; therefore, it was not considered
necessary to investigate a dosage above 1500 mg/kg/day.

- Rationale for animal assignment (if not random): The animal numbers were assigned to groups in non-sequential manner due to the need for the
functional observation battery and detailed clinical signs to be performed without knowledge of the treatment groups to which animals were
assigned.

- Section schedule rationale (if not random): The sequence in which the animals of the toxicity subgroup were killed after completion of the
observation period was selected to allow satisfactory inter-group comparison. Reproductive subgroup males were killed after the toxicity
subgroup animals and reproductive subgroup females were killed on Day 4 of lactation.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily
- Cage side observations: evidence of reaction to treatment or ill-health

DETAILED PHYSICAL OBSERVATIONS: Yes
- Time schedule: weekly
- A more detailed physical examination was performed on each animal by an observer. After removal from the home cage, animals were
assessed for physical condition and behaviour during handling and after being placed in a standard arena. During these examinations particular
attention was paid to possible signs of neurotoxicity such as convulsions, tremor and abnormalities of gait or behaviour.

ADDITIONAL DETAILED OBSERVATIONS: Yes
- Time schedule: during Week 1 daily according to the following frequency:
1. Pre-dose observation, 2.As each animal was returned to its home cage, 3.At the end of dosing each group, 4.Between 1 and 2 hours after
completion of dosing all groups and 5.As late as possible in the working day.
From Week 2 to termination daily observations were limited to 1 and 4.

BODY WEIGHT: Yes
- Time schedule for examinations: Each male and toxicity subgroup female: on the day that treatment commenced, 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:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for observations: All males and toxicity subgroup females: weekly throughout the study with the exception of the males whilst in
pairing with the reproductive subgroup females. 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.

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

OTHER:
Mating performance and fertility:
All 10 males in each group (toxicity and reproductive subgroups) were mated with the 10 reproductive subgroup females after all animals had
received 2 weeks of treatment. Pairing was on a one-to-one basis within treatment groups for up to 2 weeks, although all animals mated and were
separated within 1 week. Each morning, following pairing, the trays beneath the cages were checked for ejected copulation plugs and a wet vaginal
smear was prepared from each female and examined for the presence of spermatozoa. The day on which a sperm positive vaginal smear or at least
three copulation plugs were found was designated Day 0 of gestation. Once mating had been confirmed, males and females were separated and the
males were returned to their normal group housing.

Parturition observations and gestation length:
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.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight
gain data: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During week 5, following FOB examinations
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: toxicity subgroup animals (5 males + 5 females per dose group)
- Parameters checked: Haematocrit (Hct), Haemoglobin (Hb), Red blood cell count (RBC), Mean cell haemoglobin (MCH), Mean cell haemoglobin
concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count, Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Pit), Reticulocyte count (Retic), Blood film for abnormal morphology and
unusual cell types, including normoblasts (The most common morphological changes, anisocytosis (aniscyto), micro/macrocytosis (Microcyto/
Macrocyto), hypo/hyperchromasia (Hypochrom/Hyperchrom), Platelet clumping (PIt. Clump)), Prothrombin time (PT) and Activated partial
thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During week 5, following FOB examinations
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: toxicity subgroup animals (5 males + 5 females per dose group)
- Parameters checked: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST),
Gamma-glutamyl transpeptidase (gGT), Total Bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na),
Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic Phosphorus (Phos), Magnesium (Mg), Total protein (Total Prot), Albumin (Alb),
Albumin/globulin ratio (A/G Ratio)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: After 4 weeks of treatment
- Dose groups that were examined: Toxicity subgroup animals (5 males + 5 females per dose group)
- Battery of functions tested: Approach response, Touch response, Startle reflex (auditory), Tail pinch response, Grip strength: forelimb and
hindlimb and motor activity.
Ovaries and uterine content:
See section 7.8.1.
Fetal examinations:
See section 7.8.1.
Statistics:
For some parameters, the similarity of the data was such that analyses were not considered to be necessary.
For categorical data, the proportion of animals was analysed using Fisher's Exact test.
For continuous data, Bartlett's test was first applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome
of Bartlett's test, treated groups were then compared with the Control group, incorporating adjustment for multiple comparisons where necessary.
For bodyweight gains and organ weights, whenever Bartlett's test was found to be statistically significant, a Behrens-Fisher test was used to perform
pairwise comparisons, otherwise a Dunnett's test was used.
The following sequence of statistical tests was used for grip strength and motor activity, bodyweight change during gestation and lactation,
bodyweight and bodyweight change for offspring, food consumption and clinical pathology data:
- If 75% of the data were the same value, then a frequency analysis was applied. Treatment groups were compared using a Mantel test for a trend in
proportions and also pairwise Fisher's Exact tests for each dose group against the control.
- If Bartlett's test for variance homogeneity was not significant at the 1 % level, then parametric analysis was applied. If the F 1 test for monotonicity of dose-response was not significant at the 1 % level, Williams' test for a monotonic trend was applied. If the F 1 test was significant, Dunnett's test was
performed instead.
- If Bartlett's test was significant at the 1 % level, then logarithmic and square-root transformations were tried. If Bartlett's test was still significant, the non-parametric tests were applied. If the HI test for monotonicity of dose-response was not significant at the 1 % level, Shirley's test for a monotonic
trend was applied. If the HI test was significant, Steel's test was performed instead.

Significant differences were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
Indices:
See section 7.8.1.
Historical control data:
See section 7.8.1.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One toxicity subgroup female receiving 1500 mg/kg/day was found dead with findings that were consistent with a dosing error. This death was
therefore considered to be unrelated to treatment. A dosage-dependant increase in transient post-dosing salivation was apparent, and this was
considered more likely to be due to the palatability of the test formulations than to an effect of the test material. A dosage-dependant increase in
the number of animals with reddening of the extremities was also apparent mainly during the early stages of treatment.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The body weight gain of males at 1500 mg/kg/day appeared to be suppressed when compared with Control, such that gain between Weeks 0-5 for this group was 78% of Control. Body weight gain appeared to be unaffected for toxicity subgroup females or reproductive subgroup females
before pairing. Bodyweight gain for reproductive subgroup females receiving 1500 mg/kg/day was reduced during the first week of gestation,
after which values returned to Control-comparable levels through to termination at Day 4 of lactation. Food consumption of males at
1500 mg/kg/day appeared to be marginally lower when compared with Control. There were no effects of treatment on the amount of food
consumed by females of both toxicity and reproductive subgroups.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No effects.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No effects

ORGAN WEIGHTS (PARENTAL ANIMALS)
Bodyweight relative kidney and liver weights for toxicity subgroup females at 1500 mg/kg/day were increased, but in the absence of a histological correlate for these findings, this was of uncertain toxicological significance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
A number of toxicity subgroup males and females at 750 and 1500 mg/kg/day had thickened stomachs and associated findings. A number of
toxicity subgroup males and females at 750 and 1500 mg/kg/day exhibited horizontal bands on the incisors.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Stomachs of toxicity subgroup animals showed minimal or slight submucosal inflammation at all doses (0/5, 3/5, 4/5 and 2/5 males and 0/5, 2/5, 4/5 and 4/5 females at 0, 250, 750 and 1500 mg/kg bw respectively). It is thought that these findings may have been associated with an irritant
effect of the test formulations rather than systemic toxicity. Histological processing of the teeth that showed horizontal banding failed to detect
any involvement of areas examined suggesting that the banding was probably restricted to the enamel of the teeth.

OTHER FINDINGS (TOXICITY SUBGROUP ANIMALS)

HAEMATOLOGY:
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 CHEMISTRY:
The following changes were recorded among toxcicity subgroup males for which an effect of treatment could not be discounted: a non dosage-
dependant elevation of alkaline phosphatase levels at 750 and 1500 mg/kg/day (132 and 131% of control, resp.); reduced glucose and
phosphorous levels at 1500 mg/kg/day (79 and 82% of control, resp.); a small but dosage-dependant reduction in total protein at 750 and 1500 mg/kg/day (93 and 91% of control, resp.) with a slightly elevated albumin/globulin ratio at the top dosage (117% of control). Changes in toxicity
subgroup females were limited to a decrease in phosphorous levels at 1500 mg/kg/day (81% of control) although a non-significant increase in
alkaline phosphatase levels at 1500 mg/kg/day (122% of control) in particular suggested a similar change to that recorded in males.

NEUROBEHAVIOUR:
No effects.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
>= 1 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
VIABILITY (OFFSPRING)
No effects

CLINICAL SIGNS (OFFSPRING)
No effects

BODY WEIGHT (OFFSPRING)
No effects.

GROSS PATHOLOGY (OFFSPRING)
No effects.

HISTOPATHOLOGY (OFFSPRING)
No effects.

OTHER FINDINGS (OFFSPRING)
The percentage of males in the litters did not indicate any preferential mortality amongst either sex.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 1 500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects on reproduction/developmental toxicity (highest dose tested)

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
NOAEL: 1500 mg/kg/day for reproduction/developmental toxicity.