Hydroxylammonium chloride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 Apr - 24 May 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

(Chbb:THOM (SPF)

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: Approximately 248 g (mean)
- Housing: Animals were individually housed in type DK III stainless weel wire mesh cages (Becker & Co, Castrop-Rauxel, Germany) with a floor area about 800 cm².
- Diet: Ground Kliba 343 feed for rat/mouse/hamster (Klingenthalmühle AG, Kaiseraugst, Switzermühle, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q-water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared in intervals of not more than 4 days during the administration period by dissolving 20, 60, 200 and 400 mg of the test material in 100 mL Milli-Q ultrapure water yielding a final concentration of 1, 3, 10 and 20 mg/kg bw.

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance solutions in Milli-Q-water for a period of 4 days were carried out for a similar batch in another study (Project No.: 32C0389/8474) at the same testing facility. Samples of the test substance solutions as duplicates were sent to the analytical laboratory twice during the study period for verification of the concentrations. The test substance solutions were analyzed by photometry. As the test substance preparations were true solutions, the homogeneity had not to be proven analytically. The stability of the aqueous test substance solutions over a period of 4 days could be demonstrated. The results of the analyses of the solutions of test substance generally confirmed the correctness of the prepared concentrations. The concentration control analyses yielded values of 86.5 – 102.7% of the nominal concentrations for the 1, 3, 10 and 20 mg/kg samples.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio: 1 - 4 untreated female rats were mated with one untreated fertile male animal.
- Length of cohabitation: From about 4 p.m. to about 7.30 a.m. of 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 post coitum

Frequency of treatment:

daily

Duration of test:

Day 6 - 20 post coitum

Dose / conc.:

1 mg/kg bw/day (nominal)

Dose / conc.:

3 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

20 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a preliminary study (reference 7.8.2-1), test substance caused clear signs of maternal toxicity at 15 and 30 mg/kg bw/d in the form of a severe haemolytic anemia with a considerable enlargement of the spleen. Marginal indications of these effects occurred already at the lowest dose of 5 mg/kg bw/d. Due to the stage of development of the implants on the day when the study was terminated (Day 16 p.c.) the examination of the uterus content was very limited; taking this into consideration, no adverse effects on the fetuses occurred.
Taking into consideration the results of this study (reference 7.8.2-1), the following doses were fixed for the full-scale prenatal toxicity study in rats: 1, 3, 10 and 20 mg/kg bw.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes, all females were observed for mortality and mobidity.
- Time schedule: Twice a day on working days and once daily on weekend and on public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily and several times daily in case of clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was recorded on Days 0, 1, 3, 6, 8, 10, 13, 15. 17 and 20 p.c..

FOOD CONSUMPTION: Yes
- Food consumption was recorded on Days 3, 6, 8, 10, 13, 15, 17 and 20 p.c..

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 p.c.
- A gross pathology was performed and the organ 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

Fetal examinations:

- External examinations: At necropsy each fetus was weighed, sexed and examined macroscopically for any external findings. The sex was determined by observing the distance between the anus and the base of the genital tubercle and was later confirmed in all fetuses fixed in Bouin`s solution by internal examination. If there were discrepancies between the "external" and the "internal" sex of a fetus, the fetus was finally sexed according to the appearance of its gonads. Furthermore, the viability of the fetuses and the condition of the placentae, the umbilical cords, the fetal membranes and fluids were examined. Individual placental weights were recorded. After these examinations, approximately one half of the fetuses per dam was placed in ethyl alcohol and the other half was placed in Bouin's solution for fixation and further evaluation.
- Soft tissue examinations: After fixation in Bouin's solution, approximately one half of the foetuses of the dams of all groups was examined for any findings in the organs according to the method of Barrow and Taylor. After these examinations the relevant foetuses were discarded.
- Skeletal examinations: After fixation in ethyl alcohol, the skeletons of approximately one half of the foetuses were stained according to a modified method of Dawson. Thereafter, the skeletons of these foetuses were examined under a stereomicroscope. After these examinations the relevant foetuses were retained by litter.

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 weiqht 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.

Historical control data:

Fetal and litter incidences for skeletal and total malformations and mean percentage of affected foetuses/litter from several oral prenatal toxicity studies in Wistar rats were provided in the study report.
Please refer to Table 1 under "any other information on materials and methods incl. tables".

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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 examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute (160 and 195%, respectively) and relative (161 and 198%, respectively) spleen weights were significantly higher in females treated with 10 and 20 mg/kg bw/d when compared with the control group. The effects on the spleen were considered to be treatment-related and toxicologically relevant.

Please refer to Table 1 under "any other information on results incl. tables".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The enlargment of the spleens of all dams treated with 10 and 20 mg/kg bw/d is in-line with the distinct increases in absolute and relative spleen weights in these treatment groups. The effects on the spleen were considered to be treatment-related and toxicologically relevant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

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

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no treatment-related adverse effects on dams observed

Key result

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

A slightly increased weight of the male foetuses of the 20 mg/kg bw/d treatment group was observed. The respective mean value of 4.1 g was fully within the range of the historical control data according to the study director.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The significant higher number of male foetuses of the 10 mg/kg bw/d treatment group in comparison to the control was without any biological relevance according to the study director.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Only two external malformations (atresia ani and acaudia) were recorded for one 1 mg/kg bw/d foetus. Both malformations, which were considered to be spontaneous in nature, were also present at a low incidence in the historical control data of the breeder that were provided in the study report. The external examination of the foetuses revealed no variations in any group.
One so-called unclassified observation (placentae fused) was recorded for three foetuses of test group 1 (1 mg/kg bw/d), one foetus of test groups 2 and 3 (3 or 10 mg/kg bw/d) each and two foetuses of test group 4 (20 mg/kg bw/d). Fused placentae appear also occasionally in control animals and were therefore not associated with treatment.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Various malformations of the sternum (sternebra(e) bipartite, ossification centers dislocated), the vertebral column (vertebral body/bodies dumbbell-shaped (asymmetrical) or bipartite (asymmetrical), vertebrae fused and/or of irregular shape, vertebrae absent), the ribs (bifurcated, absent or fused) and/or the pelvic girdle (os ilium deformed) were seen in 4 out of 145 (= 2.8%) fetuses (in 3 out of 20 litters (= 15%)) of the control, in 3 out of 165 (= 1.8%) fetuses (in 3 out of 22 litters (= 14%)) of the 1 mg/kg bw/d treatment group, in 13 out of 159 (= 8.2%) fetuses (in 10 out of 23 litters (= 43%)) of the 3 mg/kg bw/d treatment group, in 14 out of 169 (= 8.3%) fetuses (in 10 out of 23 litters (= 43%) of the 10 mg/kg bw/d treatment group and in 9 out of 175 (= 5.1%) fetuses (in 5 out of 24 litters (=21%)) of the 20 mg/kg bw/d treatment group. All skeletal malformations recorded appeared without a clear dose - response relationship, but the litter incidences for total skeletal malformations and the percentages of affected fetuses/litter with overall skeletal malformations were statistically significantly increased in both intermediate dose levels (3 and 10 mg/kg bw/d) in comparison to the concurrent control group. Most of the aforementioned skeletal malformations or very similar ones, however, can be found at a comparable fetal/litter incidence in the historical control data, that were provided in the study report.

The variations elicited were related to the ribs (wavy, shortened 13th, accessory 14th or rudimentary cervical rib(s)), the sternum (sternebra(e ) of irregular shape or bipartite, accessory sternebral, the skull (epactal bone between parietal and interparietal bones), the vertebral column (accessory lumbar vertebra) and the clavicula (deformed). All skeletal variations 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. In all groups signs of skeletal retardation occurred substantiated by incomplete or missing ossification of skull (incl. hyoid) bones, vertebral column, sternebra(e) and metatarsal bones.

According to the study director, all differences between the groups in respect to skeletal retardations are without any biological relevance and do not show any dose- response relationship.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The examination of the organs of the foetuses revealed two kinds of malformations. Situs inversus was recorded for one fetus of test group 3 (10 mg/kg bw/d) and an unilobular lung was seen in one foetus of test group 4 (20 mg/kg bw/d). One of these malformations (situs inversus) is also present at a low incidence in the historical control data. The isolated and disparate nature of these findings does not suggest any treatment-related aetiology; therefore, both soft tissue malformations are considered to be spontaneous in nature.
Variations (dilated renal pelvis and/or hydroureter) were detected in all groups without any statistically significant and/or biologically relevant differences between the groups. Both findings are very common ones in the rat strain used and all respective values are fully in the range of biological variation. One so-called unclassified observation (bloody imbibition of kidney(s)) was recorded for one foetus of test groups 1, 2 and 4 (1, 3 or 20 mg/kg bw/d) each. From the historical control data it becomes obvious that this finding was already seen in control foetuses; therefore, the isolated and not dose-related occurrence of bloody imbibition of kidney (s) is assessed as an incidental finding according to the study director.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: No adverse effects on the fetuses occured at the highest tested dose level of 20 mg/kg bw.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Absolute and relative spleen weights

Dose group (mg/kg bw)		Absolute spleen weight (g)	Relative spleen weight (%)
Control	Mean	0.72	0.179
	SD	0.129	0.0278
	N	20	20
1	Mean	0.73	0.183
	SD	0.089	0.0201
	N	22	22
3	Mean	0.72	0.186
	SD	0.081	0.0201
	N	23	23
10	Mean	1.16 **	0.289 **
	SD	0.161	0.0350
	N	23	23
20	Mean	1.41 **	0.355 **
	SD	0.155	0.0339
	N	24	24

Conclusions:

The test substance bis(hydroxylammonium) sulphate showed no effects on developmental toxicity.

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

(maternal exposure until Day 15 p.c. and termination of the study on Day 16 p.c., one or two days before expected delivery is recommended in the OECD TG; the examination of the uterus content was very limited due to the stage of development of the implants, no skeletal and soft tissue alterations were examined, only ten females/dose group were exposed)

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted Jan 2001

Deviations:

yes

Remarks:

(shorter maternal exposure and earlier termination of the study than recommended in the OECD TG; limited examination of the uterus content, no skeletal and soft tissue alterations were examined, only ten females/dose group were exposed)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. K. Thomae GmbH, Biberach/Riss, Germany
- Age at study initiation: Approximately 10 - 15 weeks
- Weight at study initiation: Approximately 200 - 300 g
- Fasting period before study: At least 5 days
- Housing: Animals were individually housed in steel cages Type DK III (EBECO, Becker & Co., Castrop-Rauxel, Germany)
- Diet: Pelleted diet of Kliba-Labordiät Ratte-Maus "A" Haltung GLP 343 (Klingentalmühle AG, Kaiseraugst, Germany), ad libitum
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): Fully air-conditioned rooms
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q ultrapure water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared at least every 4 days by dissolving 100, 300 or 600 mg of the test material in 100 mL Milli-Q ultrapure water yielding a final concentration of 5, 15 and 30 mg/kg bw.

VEHICLE
- Amount of vehicle: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The homogeneity of the test substance was not determined due to the high purity of the test substance. The stability of the test substance in the vehicle of at least 4 days was confirmed during a drinking water study with the tes substance performed at the same testing facility. Furthermore, to check the accuracy of the concentration of the dosing solutions, samples of the test substance solutions as duplicates were sent to the analytical laboratory at the beginning and at the end of this study for verification of the concentrations.

Results of the analytical investigation:
100 mg/mL: 105.5, 107.8, 89.5 and 86.5 mg/100 mL
300 mg/100 mL: 314.2, 319.4, 315.0 and 314.0 mg/100 mL
600 mg/100 mL: 590.8, 607.2, 606.0 and 628.0 mg/100 mL

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: An untreated female rat was cohoused with an untreated male rat of the same breed.
- M/F ratio per cage: 1/1
- Length of cohabitation: From 4 p.m. to 7.30 a.m. of 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 post coitum

Frequency of treatment:

daily

Duration of test:

Day 6 - 16 post coitum

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Repeated dose toxicity studies with the test substance were performed at the same testing facility. In these studies, the test substance was administered via drinking water in concentrations of 25, 100, 400 and 1600 ppm for 4 weeks and in concentrations of 10, 50 and 250 ppm for 3 months, respectively. Haemolytic anaemia, metaemoglobinaemia and an increase of the liver and spleen weights with corresponding histopathological findings (such as extramedullary hematopoiesis, hemosiderin deposits and/or sinus enhancement in the spleen) were observed at concentrations ≥ 4 mg/kg bw (equivalent to 50 ppm). Based on these findings, the highest dose level for developmental testing was chosen to be 30 mg/kg bw.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes, all females were observed for mortality and mobidity.
- Time schedule: At least twice a day from monday to friday and once daily from saturday to sunday and on public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily and several times daily in case of clinical signs.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was recorded on Days 0, 1, 3, 6, 8, 10, 13, 15 and 16 p.c..

FOOD CONSUMPTION: Yes
- Food consumption was recorded on Days 3, 6, 8, 10, 13, 15 and 16 p.c..

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 16 p.c.
- A gross pathology was performed and the organ weight of liver, kidneys and spleen was recorded.
- Haematological parameters were checked: leukocytes, erythrocytes, differential blood count, reticulocytes, haemoglobin, haematocrit, mean cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thrombocytes, Heinz bodies, total haemoglobin, oxyhaemoglobin, carboxyhaemoglobin, methaemoglobin, oxygen concentration, oxygen saturation, reduced haemoglobin and oxygen capacity
- Coagulation test: Hepato Quick-Test
- Enzymes were checked: alanin-aminotransferase, aspartat-aminotransferase, alkaline phosphatase, gamma-glutamyltransferase
- Clinical chemistry parameters were checked: sodium, kalium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globuline, tryglycerides, cholesterol and magnesium

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
- Other: Position of implantation sites in uterus

Fetal examinations:

- External examinations: Yes, the weight of every foetus and placenta was recorded. The macroscopical examination for external findings (including the condition of the placentae, the umbilical cords, the fetal membranes and fluids) was only possible to a limited extent due to the early developmental stage according to the study director. Thereafter, the foetuses were discarded.

Statistics:

Dunnett test was used for food consumption, body weight, body weight gain, corrected body weight gain, clinical and hematological parameters (except of differential blood count), organ weight of liver, kidneys, spleen and gravid uterus, weight of placenta and foetus, corpora lutea, implantations, pre- and post-implantation loss, resorption and alive fetuses.
Fischer`s Exact test was used for conception rate, mortality rate (dams) and findings on fetuses.

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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):

Haemotological findings:
Compared with the control group, increased white blood cells (128 and 185% for the mid- and high-dose females, respectively) were observed in the two highest dose groups which are correlating with an increase in polymorphonuclear neutrophils (154 and 211%, respectively) and lymphocytes (122 and 170%, respectively) and in these two treatment groups and with an increase of monocytes (183%) and of the number of monoblasts in the highest dose group.
Red blood cells 79 and 72%, respectively), hemoglobin (91 and 90%, respectively) and hematocrit (90% for both dose groups) were significantly decreased in the mid- and high-dose groups. Mean corpuscular volume was significantly increased in all dose groups (103, 114 and 124%, respectively) and mean corpuscular hemoglobin (116 and 124%, respectively) was increased in the females dosed at 15 and 30 mg/kg bw when compared with the control group. In the differential blood count of the animals given 15 and 30 mg/kg bw increased polychromasia, anisocytosis, macrocytosis, normoblasts and Howell-Jolly bodies were detected. Furthermore, a dose-dependent significant increase was seen in all treatment groups and an extreme increase in Heinz bodies was observed in the mid- and high-dose females.
According to the study director, all changes in the red blood count 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.

Haemoglobin status:
Significantly decreased values for total haemoglobin (89 and 82%, respectively), oxygen content (85 and 69%, respectively) and oxygen capacity (89 and 81%, respectively) were found in the peripheral blood of the animials treated with 15 and 30 mg/kg bw compared with control group. Moreover, reduced oxyhaemoglobin (84%) and oxygen saturation (84%) and increased deoxygenated haemoglobin (122%) were detected in the highest dose group compared with control animals.
According to the study director, the changes seen in the haemoglobin status were treatment-related which were caused by a disturbance of the oxygen supply which is accociated with the anemic process.

Please refer to Table 1 for haematological findings and Table 2 for differential blood count under "any other information on results incl. tables".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Significantly increased total bilirubin concentrations (206 and 27%, respectively) were found in the serum of the mid- and high-dose females when compared with control animals. The increase in bilirubin is due to an increased rate of haemoglobin degradation as the result of the accelerated destruction of red blood cells in the anemic process and was therefore considered to be treatment-related.

Please refer to Table 1 under "any other information on results incl. tables".

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute (increase of 189 and 241%, respectively) and relative (178 and 230%, respectively) spleen weights were distinctly increased in females dosed at 15 and 30 mg/kg bw compared with the control group. Furthermore, slightly increased absolute (increase of 113%) and relative (increase of 109%) spleen weights were also noted for the low-dose females without statistical significance when compared with the control females.

The effects on absolute and relative spleen weights correlate with the effects on haemotalogical findings and are therefore treatment-related and toxicological relevant.

Please refer to Table 3 under "any other information on results incl. tables".

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The spleen of the animals of the mid- and high-dose group was considerably enlarged compared with the spleen of the control group. This effect was in correlation to the increased spleen weights and the haematological findings, thus the enlargment of the spleen was treatment-related and toxicologically relevant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

One fetus of the control group died. According to the study director, this effect was considered to be incidental and within the normal range of deviations for animals of this strain and age.

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:

effects observed, non-treatment-related

Description (incidence and severity):

One dam of the control group and one dam of the mid-dose group were not pregnant. According to the study director, this effect was considered to be incidental and within the normal range of deviations for animals of this strain and age.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

haematology

organ weights and organ / body weight ratios

Key result

Dose descriptor:

LOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

gross pathology

haematology

organ weights and organ / body weight ratios

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

not examined

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

According to the study director, the examination of the uterus content was very limited due to the stage of development of the implants on the day when the study was terminated (Day 16 p.c.), taking this into consideration, no adverse effects on the fetuses occured.

Remarks on result:

other: See remarks:

Remarks:

Due to the stage of development of the implants on the day when the study was terminated (Day 16 p.c.) the examination of the uterus content was very limited; taking this into consideration, no adverse effects on the fetuses occurred at the highest dose level of 30 mg/kg bw/d.

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 1: Summary of haematological findings and clinical chemistry

Dose group (mg/kg bw)		White blood cells (10^9/L)	Red blood cells (10^12/L)	Haemoglobin (mmol/L)	Haematocrit (L/L)	Mean corpuscular volume (fL)	Mean corpuscular heamoglobin (fmol)	Reticulocytes (‰)	Heinz Bodies (‰)	Total Haemoglobin (mmol/L)	Oxyhaemoglobin (%)	Content of oxygen bound to haemoglobin (mmol/L)	Oxygen saturation (%)	Reduced haemoglobin (%)	Oxygen capacity (mmol/L)	Total bilirubin (µmol/L)
Control	Mean	4.75	6.65	7.49	0.383	57.51	1.13	28	11	8.4	57.9	4.8	59.3	39.8	8.2	2.06
	SD	0.47	0.47	0.32	0.021	1.71	0.06	6	2	0.2	7.5	0.6	7.9	7.9	0.2	0.94
	N	9	9	9	9	9	9	9	9	9	9	9	9	9	9	9
5	Mean	4.96	6.38	7.38	0.380	59.52 *	1.16	56 **	37	8.2	54.3	4.4	55.7	43.3	8.0	2.63
	SD	0.92	0.53	0.39	0.034	0 96	0.06	12	15	0.5	5.5	0.5	5.8	5.8	0.6	0.51
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
15	Mean	6.08	5.25 **	6.88 **	0.345 **	65.79 **	1.31 **	119 **	444 **	7.5 **	54.3	4.1 **	55.9	42.8	7.3 **	4.25 **
	SD	1.33	0.43	0.29	0.022	1.68	0.06	13	31	0.4	4.4	0.4	4.6	4.5	0.4	0.62
	N	10	9	9	9	9	9	9	9	9	9	9	9	9	9	9
30	Mean	8.79 **	4.83 **	6.76 **	0.345 **	71.47 **	1.40 **	233 **	598 **	6.9 **	48.7 **	3.3 **	50.1 **	48.6 *	6.7 **	4 69 **
	SD	2.04	0.28	0.23	0.016	7.97	0.08	29	83	0.4	6.5	0.7	6.8	6.8	0.4	0.97
	N	9	10	10	10	10	10	10	10	10	10	10	10	10	10	10

Statistics: Anova + Dunnett`s test (two-sided): * p ≤ 0.05; ** p ≤ 0.01

Table 2: Morphological variations in red blood cells: Differential blood count

Dose group (mg/kg bw)	Monoblasts	Polychromasia	Anisocytosis	Macrocytosis	Normoblasts	Howell-Jolly Bodies
Control	1/9	0/9	1/9	0/9	0/9	0/9
5	2/10	0/10	1/10	0/10	0/10	0/10
15	2/9	9/9	8/9	3/9	3/9	9/9
30	7/10	9/10	10/10	10/10	7/10	10/10

Table 3: Summary of absolute and relative spleen weights

Dose group (mg/kg bw)		Absolute spleen weight (g)	Relative spleen weight (%)
Control	Mean	0.719	0.2453
	SD	0.1051	0.02696
	N	9	9
5	Mean	0.816	0.2696
	SD	0.0768	0.02104
	N	10	10
15	Mean	1.360 **	0.4369 **
	SD	0.1682	0.04886
	N	9	9
30	Mean	1.739 **	0.5655 **
	SD	0.1775	0.04590
	N	10	10

Conclusions:

The test substance bis(hydroxylammonium) sulphate showed no effects on developmental toxicity.

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

Hydroxylammonium chloride

EC Number:

226-798-2

EC Name:

Hydroxylammonium chloride

Cas Number:

5470-11-1

Molecular formula:

ClH.H3NO

IUPAC Name:

hydroxyammonium chloride

Test material form:

solid

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no treatment-related adverse effects on dams observed

Remarks on result:

other: Source: 7.8.2-1

2

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

organ weights and organ / body weight ratios

Remarks on result:

other: Source: 7.8.2-1

3

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

haematology

organ weights and organ / body weight ratios

Remarks on result:

other: Source: 7.8.2-2

4

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

gross pathology

haematology

organ weights and organ / body weight ratios

Remarks on result:

other: Source: 7.8.2-2

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

> 20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on the fetuses occured at the highest tested dose level of 20 mg/kg bw.

Remarks on result:

other: Source: 7.8.2-1

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the analogue approach, no adverse effects on developmental toxicity are considered for hydroxylammonium chloride. The NOAEL systemic was considered to be 3 mg/kg bw/day in dams and the NOAEL for developmental toxicity was considered to be > 50 mg/kg bw/day in the offspring.