Bis(hydroxylammonium) sulphate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27. Apr 1993 - 24. May 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD 414)

Cross-reference

Reason / purpose for cross-reference:

other: Range-finding study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Only limited (range finding) study with the gravidity period reduced (up to day 16 only).

Reason / purpose for cross-reference:

other: Main study

Principles of method if other than guideline:

Range finding study in which 0, 5, 15 and 30 mg hydroxylammoniumsulfate/kg body weight/day were administered by gavage to pregnant rats during organogenesis.

GLP compliance:

not specified

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. K. Thomae GmbH, Biberach/Riss
- Age at study initiation: 10-15 weeks
- Weight at study initiation: ca. 200-300 g
- Housing: single housing in mesh wire cages type DK III, EBECO, Becker &Co., Castrop-Rauxel
- Diet (e.g. ad libitum): ad libitum (Kliba diet "Ratte-Maus A Haltung GLP 343", Klingentalmühle AG, CH-4303 Kaiseraugst)
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q-purified water

Analytical verification of doses or concentrations:

yes

Remarks:

after end of experiment all concentrations were analytically verified, stability of test substance in vehicle was demonstrated before the start of the experiment.

Details on analytical verification of doses or concentrations:

after end of experiment all concentrations were analytically verified, stability of test substance in vehicle was demonstrated before the start of the experiment.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: not specified
- Length of cohabitation: 4PM until 7.30 AM of the following day
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

day 6 - 15 p.c.

Frequency of treatment:

daily

Duration of test:

11 days

Remarks:

Doses / Concentrations:
5, 15, 30 mg/kg bw/d (ca. 2, 6.1, and 12.3 mg base/kg bw/d)
Basis:
nominal conc.

No. of animals per sex per dose:

9 or 10 pregnant rats were applied per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on data available from repeated dose toxicity studies and the results observed (see IUCLID section 7.5)
- Rationale for animal assignment (if not random): random

Maternal examinations:

CAGE SIDE OBSERVATIONS: At least once per day, if symptoms were observed several observations per day


DETAILED CLINICAL OBSERVATIONS: No data


BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 1, 3, 6, 8, 10, 13, 15 and 16 post coitum


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 16
- Hematology: Leukocytes, Erythrocytes, Hemoglobin, Hematocrit, MCV, Hemoglobin content per erythrocyte, MCH, thrombocytes, differential hematology, reticulocytes, Heinz bodies
- Hemoglobin status: total hemoglobin, oxihemoglobin, carboxihemoglobin, methemoglobin, oxygen content, oxygen saturation, reduced hemoglobin, oxygen capacity
- coagulation: Hepato Quick test
- enzymes: Alanin aminotransferase, aspartate aminotransferase, alkalic phosphatase, gamma glutamyltransferase
- clinical chemistry: sodium, potassium, chloride, phosphate, calcium, urea, creatinin, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium
- Gross pathology: liver weight, kidney weight, spleen weight, unopened uterus weight, number of corpora lutea, number of implantations early resoptions, situation of implants in uterus

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes. The number of resorptions was determined (not further specified).
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: yes
- Number of implantations: yes
- Number of early resorptions: yes
- Number of late resorptions: No data

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Fetal weights were determined

Statistics:

Dunnett test (food consumption, body weights, body weight changes, corrected body weight change, clinical chemistry and hematology, organ weights, placenta weights and fetus weights, corpora lutea, impantations, pre- and post-implantation loss, resorptions, living fetuses
Fisher's exact test: conception rate, mortality rate (maternal), fetal findings

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Increased white blood cells were observed in the two highest dose groups which are correlating with an increase in polymorphonuclear neutrophils and lymphocytes in these two treatment groups and with an increase in monocytes and monoblasts in the highest dose group.
Red blood cells, hemoglobin and hematocrit were statistically significantly decreased in the mid- and high-dose groups. Mean corpuscular volume (MCV) was statistically significantly increased in all dose groups and mean corpuscular hemoglobin (MCV) was increased in test groups
2 and 3 (15 mg/kg body weight and 30 mg/kg body weight).
In the differential blood count of the animals given 15 mg/kg body weight and 30 mg/kg body weight increased polychromasia, anisocytosis, makrocytosis, normoblasts and Howell-Jolly bodies were detected. Furthermore, a dose-dependent, statistically significant increase in
reticulocytes was seen in all treatment groups and an extreme increase in Heinz bodies was observed in the mid- and high-dose.
All changes in the red blood picture are signs of a severe anemia with Heinz body-formation caused by the test substance. The findings observed in the white blood picture are also treatment-related and might be a consequence of the anemic process.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significantly increased total bilirubin concentrations were found in the serum of the intermediate and high-dose rats (15 or 30 mg/kg body weight/day). The increase in bilirubin is due to an increased rate of hemoglobin degradation as the result of the accelerated destruction of red blood cells in the anemic process.
The other clinicochemical examinations revealed no treatment-related changes.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absoiute and relative spleen weights were distinctly increased and the spleens were considerably enlarged in the intermediate (15 mg/kg body weight/day) and the high dose group (30 mg/kg body weight/day). Furthermore, slightly, but statistically insignificantly increased spleen weights were also noted for the low dose rats (5 mg/kg body weight/day). These findings are attributed to the test substance administration and are a consequence of the hemolytic anemia.
The weights of the gravid uteri were not influenced by the administration of the test substance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A spontaneous necropsy finding (hydrometra) occurred in one control dam, which did not become pregnant.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Hemoglobin status:
Statistically significantly decreased values for total hemoglobin, oxygen content and oxygen capacity were found in the peripheral blood of the animals of test groups 2 and 3 (15 mg/kg body weight and 30 mg/kg body weight).
Moreover, reduced oxyhemoglobin and oxygen saturation and increased deoxygenated hemoglobin were detected in the highest dose group.
The changes seen in the hemoglobin status are substance related, too. They are caused by a disturbance of the oxygen supply which is associated with the anemic process.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate varied between 90% (control group and test group 2) and 100% (test group 1 and 3).

Details on maternal toxic effects:

There were no substance-related and/or statistically significant differences between the groups in conception rate, the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and postimplantation losses, the number of resorptions and viable fetuses. The differences evident are considered to be incidental and within the normal range of deviations for animals of this strain and age.

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Abnormalities:

effects observed, treatment-related

Localisation:

other: blood

Description (incidence and severity):

mid and high dose:
- increase in white blood cells, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), polymorphonuclear neutrophils, lymphocytes, monocytes, monoblasts, polychromasia, anisocytosis, makrocytosis, normoblasts, Howell-Jolly bodies, reticulocytes, Heinz bodies, deoxygenated hemoglobin and total bilirubin
- decrease in red blood cells, hemoglobin, hematocrit, oxyhemoglobin, oxygen content, oxygen saturation and oxygen capacity
- statistically significantly increased absolute and relative spleen weights with considerably enlargement of the spleen
==> severe hemolytic anemia with considerable enlargement of the spleen.

low dose shows marginal indications of these effects with increased MCV and reticulocytes as well as marginally increased absolute and relative spleen weights.

Fetal body weight changes:

no effects observed

External malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Gross macroscopic examination of fetuses revealed no teratogenic effects.

Dose descriptor:

NOAEL

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Clinical chemistry and hematology:

30 mg/kg body weight:

Increase in white blood cells, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), polymorphonuclear neutrophils, lymphocytes, monocytes, monoblasts, polychromasia, anisocytosis, makrocytosis, normoblasts, Howell-Jolly bodies, reticulocytes, Heinz bodies, deoxygenated hemoglobin and total bilirubin.

Decrease in red blood cells, hemoglobin, hematocrit, oxyhemoglobin, oxygen content, oxygen saturation and oxygen capacity.

 

15 mg/kg body weight:

Increase in white blood cells, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), polymorphonuclear neutrophils, lymphocytes, polychromasia,

anisocytosis, makrocytosis, normoblasts, Howell-Jolly bodies, reticulocytes, Heinzbodies and total bilirubin.

Decrease in red blood cells, hemoglobin, hematocrit, oxygen content and oxygen capacity.

 

5 mg/kg body weight:

Increase in mean corpuscular volume (MCV) and reticulocytes.

Spleen weights:

	Spleen weights
Dose	absolute [g]	relative [% final body weight]
0 mg/kg bw/day	0.72 (100%)	0.245 (100%)
5 mg/kg bw/day	0.82 (114%)	0.270 (110%)
15 mg/kg bw/day	1.36** (189%)	0.437** (178%)
30mg/kg bw/day	1.74** (242%)	0.566** (231%)

**: p<0.01

From the results of this preliminary study it was obvious, that hydroxylammoniumsulfate caused clear signs of maternal toxicity at 15 and 30 mg/kg body weight in the form of a severe hemolytic anemia with a considerable enlargement of the spleen. Marginal indications of these effects occurred already at the lowest dose, 5 mg/kg body weight/day.

Due to the stage of development of the implants on the day of study termination the examination of the uterus content was very limited. Taking this into consideration, no adverse effects on the fetuses occurred.

 

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Bis(hydroxylammonium) sulphate

EC Number:

233-118-8

EC Name:

Bis(hydroxylammonium) sulphate

Cas Number:

10039-54-0

Molecular formula:

H3NO.1/2H2O4S

IUPAC Name:

bis(hydroxyammonium) sulfate

Details on test material:

- Name of test material (as cited in study report): Hydroxylammoniumsulfate (solid salt)
- Physical state: solid, crystals/white
- Analytical purity: > 98.4%
- Lot/batch No.: continuous production of April 4, 1991
- Storage condition of test material: refrigerator, dry

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: 76 99 days
- Weight at study initiation: 248 g (average)
- Housing: singly in type DK III stainless steel wire mesh cages (floor area about 800 cm2) .
- Diet: ground Kliba 343 feed rat/mouse/hamster supplied by KLINGENTALMUEHLE AG, Switzerland, ad libitum
- Water: tap water quality from water bottles, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24°C
- Humidity (%): 30 - 70 %
- Photoperiod: 12 hrs dark / 12 hrs light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Milli-Q-water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the test substance:
Analytical determinations of the purity of the test substance itself were carried out before the beginning of the study (method: potentiographic titration). The stability of the test substance was proven by reanalysis.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1-4
- Length of cohabitation: from about 16.00 hours to about 7.30 hours on the following day.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

day 6 - 15 p.c.

Frequency of treatment:

10x by gavage

Duration of test:

20 days

Doses / concentrations

Remarks:

Doses / Concentrations:
1, 3, 10, 20 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

25 (females)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose selection: In a pre-study it became obvious that HAS caused clear signs of maternal toxicity at 30 and 15 mg/kg bw/day (hemolytic anemia).
Therefore, 20 mg was chosen as the upper dose.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were examined for clinical symptoms at least once a day, or more often when clinical signs of toxicity were elicited.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on days 0, 1, 3, 6, 8, 10, 13, 15, 17 and 20 p.c.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: gross pathology, weight of spleen was recorded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Furthermore, calculations of conception rate and preand postimplantation losses were carried out.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

The DUNNETT-Test was used for a simultaneous comparison of several dose groups with the control. The hypothesis of equal means was tested. This test was performed two-sided and was used for the statistical evaluation of the following parameters: Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), weight of the uterus before it was opened, spleen weights (absolute and relative), number of corpora lutea, number of implantations, number of resorptions and number of live fetuses; proportion of preimplantation loss, postimplantation loss, resorptions and live fetuses in each litter; litter mean fetal body weight and litter mean placental weight.

FISHER's Exact Test was used for a pairwise comparison of each dose group with the control for the hypothesis of equal proportions. This test was performed one-sided and was used for female mortality, females pregnant at terminal sacrifice and the number of litters with fetal findings.

The WILCOXON-Test was used for a comparison of each dose group with the control for the hypothesis of equal medians. This test was performed one-sided and was used for the proportion of fetuses with malformations, variations, retardations and/or unclassified observations in each litter.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Absolute and relative spleen weights were statistically significantly higher in the 10 and 20 mg/kg bw groups than in the control group. In the 10 mg/kg bw group the spleen weights were approx. 60% higher than the respective control values, whereas the spleen weights of the high dose group dams were nearly twice as high as in the control group. The increased spleen weights are well known substance induced effects. At the lower dose levels (1 or 3 mg/kg body weight) absolute and relative spleen weights were similar to the control weights.

At necropsy the enlargement of the spleens of all dams of test groups 10 and 20 mg/kg body weight/day is in-line with the distinct increases in absolute and relative spleen weights in these groups.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

3 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
All signs of embryo-/ fetotoxicity and substance-related teratogenicity, malformations recorded, appeared without a clear dose-response relationship, can be found in a similar frequency in the historical control data and/or the differences between the groups are without biological relevance.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

>= 20 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

In the dams an enlargement of the spleens and a dose related statistically significant increase in absolute and relative spleen weights was revealed at dosages of 10 and 20 mg/kg bw/day. No substance-related effects on dams were reported for the lower dose groups. There were no substance-related differences between the groups in conception rate, the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and of viable foetuses. Mean fetal body weight of the dosed groups did not differ from that of the control. Examination of foetuses did not reveal any signs for substance related abnormalities.

 

From this study a NOAEL for maternal toxicity of 3 mg/kg bw/d and a NOAEL for embryo-/fetotoxicity of 20 mg/kg bw/d can be derived.