Hydroxylammonium chloride

Endpoint summary

Description of key information

NOAEL (28 days, rat): 7.77 mg/kg bw/d

RA from source substance bis(hydroxyammonium) sulfate (CAS 10039-54-0)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

12 females and 6 males received daily for 6 days/week a supplement of the test substance, which was added to milk as a solution over a period of 178 days.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: About 3 weeks old
- Weight at study initiation: Females: 45 - 59 g; Males: 35 - 59 g
- Housing: The animals were individually housed.
- Diet: Standard diet consisting of casein (20%), dry yeast (15%), starch (55%), salt mixture (5%) and lucerne meal (5%), ad libitum. In addition, the rats were frequently fed raw carrot, lettuce or apple.

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was added to milk as a solution (pH 5.2) partly neutralized with sodium hydroxide.

PREPARATION OF THE DOSING SOLUTION
- Rate of preparation of the dosing solution: The solution for the drinking study was prepared daily.
- Mixing appropriate amounts with: The doses of the test substance were adjusted to be proportional to the weight of the rats. The rats with an average body weight of 30 - 70 g received 10 mg of the test substance (corresponding to 333 - 142 mg/kg bw/d and a mean value of 237.5 mg/kg bw/d), those with an average weight of 70 - 110 g received 20 mg of the test substance (corresponding to 285 - 181 mg/kg bw/d and a mean value of 233 mg/kg bw/d), those with an average weight of 110 - 150 g received 30 mg of the test substance (corresponding to 272 - 200 mg/kg bw/d and a mean values of 236 mg/kg bw/d) and the rats with an average weight > 150 g received 40 mg of the test substance (corresponding to 266 mg/kg bw/d).

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

178 days

Frequency of treatment:

Daily, 6 days/week

Remarks:

range: 233 - 266 mg/kg bw/d

No. of animals per sex per dose:

12 females and 6 males

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

BODY WEIGHT: Yes, the body weight was recorded at the beginning and after 5 months.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, after 178 days the rats were killed with chloroform, frozen and kept at -20 °C for up to 30 days, after which they were dissected. The rats were kept wrapped up so that they could not lose water and were dissected after partial thawing. The organs were examined macroscopically, and the following organs were weighed: heart, lungs, liver, spleen, kidneys, adrenals and thyroid.

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):

not examined

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):

The relative spleen weights were distinctly increased in treated females (1.645%) and males (1.56%) when compared with control females (0.469%) and males (0.316%), respectively. The relative thyroid weights were considerably reduced in females (0.038%) and males (0.032%) compared with control females (0.065%) and males (0.055%), respectively.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A considerable development of the spleen and a marked reduction of the thyroid were observed in the animals of the treatment group. These findings correlate with the distinct increase of the relative spleen weights and with the strong induction of the relative thyroid weights.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

LOAEL

Effect level:

233 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

233 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

spleen

thyroid gland

Treatment related:

yes

Conclusions:

CLP: STOT RE 2, H373 (Annex VI harmonized classification)

The registrant follows the harmonised classification, thus the available data with the test substance meet the classification criteria according to Regulation (EC) 1272/2008.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

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:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acc. to guidelines, but preliminary study with technical shortcomings concerning definite dosing.

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

7.77 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

by calculation based on the NOAEL of 3.29 mg/kg bw/day using a molecular weight factor of 2.36 (MW: 164.1/69.5)

Sex:

male

Basis for effect level:

other: No treatment-related adverse effects observed in the 25 ppm dose group

Key result

Dose descriptor:

NOAEL

Effect level:

26.77 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

by calculation based on the NOAEL of 11.34 mg/kg bw/day using a molecular weight factor of 2.36 (MW: 164.1/69.5)

Sex:

female

Basis for effect level:

other: No treatment-related adverse effects observed in the 100 ppm dose group

Key result

Dose descriptor:

LOAEL

Effect level:

24.35 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

by calculation based on the LOAEL of 10.32 mg/kg bw/day using a molecular weight factor of 2.36 (MW: 164.1/69.5)

Sex:

male

Basis for effect level:

other: Treatment-related adverse effects on spleen and blood parameters observed in the 100 ppm dose group

Key result

Dose descriptor:

LOAEL

Effect level:

102.09 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

by calculation based on the LOAEL of 43.26 mg/kg bw/day using a molecular weight factor of 2.36 (MW: 164.1/69.5)

Sex:

female

Basis for effect level:

other: Treatment-related adverse effects on spleen and blood parameters observed in the 400 ppm dose group

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

24.35 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

blood

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Based on the pathological findings in the spleen and the adverse effects on blood parameters found with the source substance similar effects are estimated for the target substance and thus the test substance is classified as STOT-RE 2, H373.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

7.77 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Based on a WoE approach, available information comprises adequate and reliable studies with the test substance itself and with a reference substance. Read-across is justified based on structural similarities and similar chemical behaviour. The selected studies are thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

System:

haematopoietic

Organ:

blood

spleen

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for read-across

There are data available regarding repeated dose toxicity for hydroxylammonium chloride (CAS 5470-11-1). In addition, read-across from an appropriate substance is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements, defined in Regulation (EC) No 1907/2006, Annex VIII, 8.6. Read-across is justified based on structural similarities and similar chemical behaviour. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

CAS 5470-11-1

There is an oral 178-day drinking repeated dose toxicity study in rats with the test substance hydroxyl ammonium chloride (CAS 5470-11-1) available. Male and female rats were administered 10, 20, 30 and 40 mg of the test substance in milk as solution to drink in dependence of the average body weight corresponding to a dose of approximately 233 - 266 mg/kg bw/d). No mortality and clinical signs or effects on body weight or body weight gains were observed during treatment. Gross necropsy revealed abnormalities relating to the organ size of spleen and thyroid. A considerable development of the spleen and a marked reduction of the thyroid were observed in the animals of the treatment group. These findings correlated with the distinct increase of the relative spleen weights and with the strong induction of the relative thyroid weights, which could be observed in male and female rats when compared to the control group. Under the conditions of this study and based on the toxicological endpoints evaluated, the LOAEL was considered to be 233 mg/kg bw/day for male and female rats.

CAS 10039-54-0

In addition, a study with the appropriate read-across substance bis(hydroxyammonium) sulfate (CAS 10039-54-0) was taken into account for hazard assessment. Male and female Wistar rats were administered 25, 100, 400 and 1600 ppm (equivalent to 2.77, 11.34, 43.27 and 158.73 mg/kg bw/day in females and 3.29, 10.32, 43.02 and 149.63 mg/kg bw/day in males, respectively, recalulated based on available data on weekly body weights and weekly water consumption) of the test substance in drinking water for 4 weeks. In summary, hemolytic anemia and splenomegaly were the prominent manifestations of toxicity, in particular in the 1600 and 400 ppm dose groups corresponding with cyanosis, changes in red blood parameters (enhanced levels of methaemoglobin, Heinz bodies and a shift in blood cell pattern, e.g. increase in immature forms of red blood cells - reticulocytes -, and a rise in leukocytes: neutrophile and eosinophile granulocytes, lymphocytes, and monocytes), changes in the biochemical composition of the plasma and relevant toxic effects in spleen, liver and kidneys. Increased decomposition of erythrocytes was seen as hemosiderin deposits and iron pigment deposition in these organs. Further, in Kupffer cells erythrophagocytosis was observed. Compensating effects were revealed in the spleen and liver as extramedullary hematopoiesis. Congestion of the spleen, enlargement of spleeny sinus, splenomegaly and increased organ weight of the spleen were caused by immature erythrocytes. Reticuloid hyperplasia and necrosis in bone marrow which were seen only in males at 1600 ppm, may be a possibly indication of bone marrow damage.

Under the conditions of this study and based on the toxicological endpoints evaluated, the NOAEL for systemic effects was 25 ppm (equivalent to 3.29 mg/kg bw/day) in males and 100 ppm (equivalent to 11.34 mg/kg bw/day) in females. Based on the available information from the EU Risk Assessment Report on Bis(hydroxylammonium)sulphate (CAS 10039-54-0), the source (CAS 10039-54-0) and the target substance (CAS 5470-11-1) are completely dissociated in aqueous media in hydroxylammonium cations and the respective anions. The hydroxylamine moiety represents the toxic species of both compounds and is responsible for the toxic effects (EU RAR, 2008). Therefore the different molecular weight was taken into account and the obtained NOAEL and LOAEL of the present study were recalculated using a molecular weight factor of 2.36 (MW: 164.1/69.5). In conclusion, the NOAEL for systemic effects were considered to be 7.77 mg/kg bw/day in males and 26.76 mg/kg bw/day for females.

References

EU RAR (2008): EU Risk Assessment Report on Bis(hydroxylammonium)sulphate (CAS 10039-54-0), May 2008

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to hydroxylammonium chloride, data will be generated from information on reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

 

Applying the RA-A approach, the available data on oral repeated dose toxicity with the source substance bis(hydroxyammonium) sulfate (CAS 10039-54-0) meet the criteria for classification according to Regulation (EC) No 1272/2008 and therefore the target substance Hydroxylammonium chloride (CAS 5470-11-1) will be classified as STOT RE Cat. 2 (H373). Spleen and blood were identified as primary target organs.