Ammonium sulphate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2001-12-28 to 2002-10-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Please refer to attached "Read-across justification" in IUCLID section 13.
In accordance to Regulation (EC) No 1907/2006 Annex XI 1.5. testing does not appear scientifically necessary if a read-across approach can be performed to common breakdown products. Ammonium sulfate dissociates into NH4+ and sulfate ions. Sulfate is a normal body and nutritional component and is regulated within the body. NH4+ ions are immediately transformed into urea by the liver and do not exist in the blood in relevant amounts unless in case of liver failure.

Cross-reference

Reason / purpose for cross-reference:

read-across: supporting information

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Justification for type of information:

Please refer to attached "Read-across justification" in IUCLID section 13.
In accordance to Regulation (EC) No 1907/2006 Annex XI 1.5. testing does not appear scientifically necessary if a read-across approach can be performed to common breakdown products. Ammonium sulfate dissociates into NH4+ and sulfate ions. Sulfate is a normal body and nutritional component and is regulated within the body. NH4+ ions are immediately transformed into urea by the liver and do not exist in the blood in relevant amounts unless in case of liver failure.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

low numbers of animals per dose group, only females were treated, no fertility indices were measured.

GLP compliance:

not specified

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Sex:

male/female

Basis for effect level:

other: Based on the inflammatory/degenerative stomach changes recorded at histopathological examination and extending to the lowest dosage.

Remarks on result:

not determinable

Remarks:

no NOAEL identified, CAS 7783-28-0

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

1 500 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Highest dose level applied.

Remarks on result:

other: CAS 7783-28-0

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Generation:

F1

Effect level:

1 500 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Highest dose level applied.

Remarks on result:

other: CAS 7783-28-0

Reproductive effects observed:

not specified

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

adopted 1996-03-22

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
male and CD rats, Crl:CD (TM) (SD)IGS BR strain
- Source: Charles River (UK) Limited, Margate, Kent England
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: Males: 298 - 386 g; Females: 191 - 263 g
- Housing: singly in RB3 modified cages
- Diet (e.g. ad libitum): Rat and Mouse No. 1 Maintenance Diet (Special Diets Services Ltd., Witham, Essex, England)
- Water (e.g. ad libitum): tap water from public supply
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 23 °C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2001-12-12 To: 2002-02-10 (necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Dosing volume: 10 ml/kg bw for each group

Details on mating procedure:

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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration during Weeks l, 4 and 6 of the study were analysed for test material content and found to be satisfactory.

Duration of treatment / exposure:

Exposure period: not clearly defined
Premating exposure period (males): 2 weeks
Premating exposure period (females): 2 weeks
Duration of test: not clearly defined
See freetext below

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males and 5 females per group (toxicity subgroups);
5 males and 10 females per group (reproductive subgroups)

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly
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.

BODY WEIGHT: Yes
- Time schedule for examinations: Each male and toxicity subgroup female was weighed on the day that treatment commenced, weekly thereafter, and at necropsy. Reproductive subgroup females were weighed 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 the males and toxicity subgroup females was recorded weekly throughout the study with the exception of the males whilst in pairing with the reproductive subgroup females. Food consumption for the reproductive subgroup females was recorded 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: No data

OTHER:

FUNCTIONAL OBSERVATION BATTERY
All toxicity subgroup animals were subjected to FOB evaluation on one occasion following 4 weeks of treatment. Animals were not necessarily all tested on the same day but the number of animals was balanced across the groups on each day of testing. FOB examinations included:
- Sensory reactivity and grip strength
- Motor activity

HAEMATOLOGY AND BLOOD CHEMISTRY
During Week 5, following the FOB examinations, blood samples were obtained from the orbital sinus of all animals in the toxicity subgroup, following overnight deprivation of food. Samples were collected under light general anaesthesia (using Isoflurane) following overnight deprivation of food.
Parameters examined:
Haematocrit, Haemoglobin, Red blood cell count, Mean cell haemoglobin, Mean cell haemoglobin concentration, Mean cell volume, Total white cell count, Differential WBC count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelet count), Reticulocyte count, Blood film, Prothrombin time, Activated partial thromoblastin time
Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyl transpeptidase, Total Bilirubin, Urea, Creatinine, Glucose, Total cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphorus, Magnesium, Total protein, Albumin

Litter observations:

All offspring were examined at approximately 24 hours after birth (Day 1) and the number of offspring born (live and dead) was recorded. Live offspring were individually identified within each litter by toe tattoo and any clinical observations were recorded.
Litters were observed daily for evidence of abnormal appearance or behaviour. Daily records were maintained of mortality and consequent changes in litter size. Where practical, any offspring found dead were subjected to a macroscopic examination as soon as possible. The offspring were sexed at individual weighing on Day I and Day 4 of age.

Postmortem examinations (parental animals):

GROSS PATHOLOGY
Toxicity subgroup animals were sacrificed during week 6 of treatment. The following tissues were retained, weighed and examined:
adrenals, aorta, brain, caecum, colon, duodenum, epididymis, eyes, heart, ileum, jejunum, kidneys, liver, lungs including bronchi, lymph nodes (mandibular, mesenteric, regional to masses), mammary area, oesophagus, optic nerves, ovaries, pancreas, pituitary, prostrate, rectum, salivary glands, sciatic nerves, seminal vesicles, skin, spinal cord, spleen, sternum with bone marrow, stomach, testes, thymus, thyroid with parathyroid, trachea, urinary bladder, uterus with cervix, vagina.

Reproductive subgroup males were killed after the toxicity subgroup animals. The testes, epididymides, seminal vesicles with coagulating gland and prostate were retained for each animal. Reproductive subgroup 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. Abnormal tissues were also retained.

HISTOPATHOLOGY
The tissues mentioned above from control and high dose group animals of the toxicity subgroups were examined histopathologically. Histology for reproductive subgroup animals was restricted to the retained reproductive organs and abnormalities observed at macroscopic necropsy.

Postmortem examinations (offspring):

Offspring of the reproductive sub group females were killed on Day 4 of age and subjected to a macroscopic necropsy.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Treatment at all dosages was well tolerated and there were no treatment-related deaths.
A dosage dependent increase in transient post-dosing salivation was apparent, which was considered to be due to the palatability of the test formulations rather than toxicity. A dosage-dependent 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)
Body weight gain and food consumption of males at 1500 mg/kg bw/day appeared to be suppressed when compared with the control group, such that gain between weeks 0-5 for this group was 78% of controls. The body weight gain for reproductive subgroup females receiving 1500 mg/kg bw/day was reduced during the first week of gestation, after which the values returned to levels comparable with the control.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating performance and fertility were unaffected by treatment, and parental treatment had no apparent effect on the offspring to day 4 of age.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Relative kidney and liver weights for females at 1500 mg/kg bw/day were greater than in the control group, but there were no histological changes associated.

GROSS PATHOLOGY, HISTOPATHOLOGY (PARENTAL ANIMALS)
A number of treated animals at 750 and 1500 mg/kg bw/day exhibited horizontal banding on the incisors at necropsy; histological processing of these tissues failed to detect any change in the areas examined suggesting that the banding was restricted to the enamel of the teeth. The only histological findings related to treatment were the inflammatory/degenerative stomach changes in all treated groups that were considered likely to have arisen due to an irritant effect of the test formulations.

OTHER FINDINGS (PARENTAL ANIMALS)

HAEMATOLOGY, CLINICAL CHEMISTRY
Some treatment-related effects on hematology were evident (reduction in activated partial thromboplastin time for males at 750 and 1500 mg/kg bw/day, a non dosage-dependent elevation of alkaline phosphatase levels at 750 and 1500 mg/kg bw/day, reduced glucose and phosphorous levels at 1500 mg/kg bw/day, a dosage-dependent reduction in total protein at 750 and 1500 mg/kg bw/day with a slight elevated albumin/globulin ratio at the top dosage. Changes in females were limited to a decrease in phosphorous levels and a non-significant increase in alkaline phosphatase level at
1500 mg/kg bw).

BEHAVIOUR
There were no changes apparent at behavioral testing.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Sex:

male/female

Basis for effect level:

other: Based on the inflammatory/degenerative stomach changes recorded at histopathological examination and extending to the lowest dosage.

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Results: F1 generation

Details on results (F1)

Parental treatment had no apparent effect on the offspring to day 4 of age.

Effect levels (F1)

1

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

1 500 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Highest dose level applied.

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Generation:

F1

Effect level:

1 500 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Highest dose level applied.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Tabular Summary of effects on reproduction /development

Observations Dosage (mg/kg/day)	0 (ctr)	250	750	1500
Pairs started (N)	10	10	10	10
Females showing evidence of copulation (N)	10	10	10	10
Females achieving pregnancy (N)	9	10	10	10
Conceiving days 1 -5 (N)	9	10	10	10
Conceiving days 6 -14 (N)	0	0	0	0
Pregnancy=21.5 days (N)	0	0	0	1
Pregnancy=22 days (N)	6	9	8	4
Pregnancy=22.5 days (N)	3	0	2	4
Pregnancy=23 days (N)	0	1	0	1
Dams with live young born (N)	9	10	10	10
Dams with live young at day 4 pp (N)	9	10	10	10
Implants/dam (mean)	15.7	15.7	14.1	15.4
Live pubs/dam at birth (mean)	14.8	14.6	12.7	14.0
Live pubs/dam at days 4 (mean)	14.6	14.3	12.7	14.0
Sex ratio (%m) at birth (mean)	54.2	52.7	50.0	48.5
Sex ratio (m/f) at day 4 (mean)	54.2	53.0	50.0	48.5
Male pub weight at birth (mean)	6.4	6.3	6.6	6.3
Male pub weight at day 4 (mean)	8.7	8.5	9.2	8.7
Female pup weight at birth (mean)	5.9	6.0	6.1	6.0
Female pup weight at day 4 (mean)	8.2	7.9	8.6	8.4
Abnormal Pups: Necropsy finding unremarkable
Loss of offspring
Post implantation survival index	95.2	93.5	90.0	94.8
Live birth index	99.3	99.4	100.0	95.9
Viability index	98.6	98.2	100.0	100.0