Bis(hydroxylammonium) sulphate

Endpoint summary

Description of key information

Acute toxicity oral:

Rat: LD50 = 642 mg/kg bw

Cat: LD50 > 50 and < 200 mg/kg bw (females) and > 200 mg/kg bw (males) Dermal Rat: LD50 > 500 mg/kg bw

Acute toxicity inhalation:

no mortality was observed in IHT.

Acute toxicity dermal:

Rabbit: LD50 > 1000 mg/kg bw (semiocclusive test conditions)

Rabbit: LD50 > 100 and < 500 mg/kg bw (occlusive test conditions)

Rabbit: LD50 > 400 mg/kg bw

Rabbit: LD50 > 1500 and < 2000 mg/kg bw (semiocclusive test conditions)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Principles of method if other than guideline:

BASF-Test: In principle, the methods described in the OECD Guideline 401 were used. Young adult laboratory rats were purchased from breeder. Several groups of 10 rats per sex and dose were treated simultaneously by gavage with preparations of the test substance in suitable vehicle. The concentrations of these preparations were used to achieve comparable volumes per kg body weight. Group-wise documentation of clinical signs was performed over the 14 day study period. The LD50 value was estimated on the basis of the observed mortalities.

GLP compliance:

no

Remarks:

GLP was not compulsory at the time the study was conducted.

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Gassner
- Average weight at study initiation: males: 219 g, females: 173 g

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Concentration in vehicle: 16 % (1600 mg/kg bw), 10 % (1250 and 1000 mg/kg bw), 8 % (800 mg/kg bw), 4 % (400 mg/kg bw), 2 % (200 mg/kg bw) in Aqua dest.

Doses:

200, 400, 500, 640, 800, 1000, 1250, 1600 mg/kg bw

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 7 days
- Frequency of observations: Animals were observed and examined for clinical signs of toxicity during the first hour following application, after 4 and 5 hours and further daily on working days.
- The body weights of the individual animals were gathered prior to application of the test material.
- Necropsy of survivors performed: Deceased animals and those sacrificed at the end of the observation period (on day 7 after dosing) were necropsied.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

642 mg/kg bw

Mortality:

Mortality ratio:

Dose
(mg/kg bw) - 1 hour - 24 hours - 48 hours - 7 days
-----------------------------------------------
1600 - 20/20 - 20/20 - 20/20 - 20/20
1250 - 17/20 - 18/20 - 18/20 - 18/20
1000 - 12/20 - 15/20 - 15/20 - 15/20
800 - 07/20 - 10/20 - 10/20 - 10/20
640 - 12/20 - 13/20 - 13/20 - 13/20
500 - 03/20 - 06/20 - 06/20 - 06/20
400 - 00/20 - 01/20 - 01/20 - 01/20
200 - 00/20 - 00/20 - 00/20 - 00/20

Clinical signs:

other: Observed Symptoms: 200 mg/kg: --------- Immediately after application of the test material: tremor, abdominal crouched down position, dyspnoe; without any findings after 3 days 400 - 800 mg/kg: -------------- Immediately after application of the test m

Gross pathology:

Organpathology (animals that were sacrificed 7 days after administration of the test material):
dark blue to violet or -black discolouration and enlargement of the spleen. The max. weight of the spleen was 2.0 g.
No detailled gross pathology findings were available from those animals taht died during the study period.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study with adaptations according to the question considered, good and detailed documentation.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

yes

Remarks:

The test material was tested on skin under semiocclusive- as well as under occlusive test conditions.

GLP compliance:

yes

Test type:

standard acute method

Limit test:

no

Species:

other: rabbit and rat

Strain:

other: New Zealand White and Fisher-344

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: rabbits: 2.1 to 3.4 kg; rats: 162 to 182 g
- Housing: individually in single unit, suspended steel cages
- Diet: Purina Rodent Chow 5001 (rats) and Purina High Fiber Diet 5321 (rabbits), ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature : 20 °C for rabbits and 23.3 °C for rats
- Humidity: 50 % mean value
- Photoperiod: 12 hrs dark / 12 hrs light

Type of coverage:

other: occlusive and semiocclusive

Vehicle:

other: distilled water

Details on dermal exposure:

Exposure methods
One day prior to application of the test and control materials, each animal was prepared for dosing by clipping the hair from its back and sides using
a Oster Model AS small-animal clipper. A No. 10 blade was used to remove the long hair from the rabbits and a No. 40 blade was used to remove the remaining short hair. Onlv the No. 40 blade was used on the rats. No less than 10% of the body surface was clipped.
Rabbitswere exposed for 24 hr to a single topical application of test material moistened with an equivalent weight of distilled water. The test material was covered with either a 4 x 4-in. porous gauzes patch opened to cover a 4 x 8 in. area of skin or a plastic cover. The plastic cover consisted of either a 25-mm Hill Top Chamber from which the cotton swatch was removed or a medium-size disposable weighing boat, the depth of which was reduced by trimming off part of the wall . The weighing boat was used for dose levels of 0.5 g/kg or greater. Both the gauze and plastic covers were held in place with Blenderm surgical tapee The entire trunk of the animal was wrapped with Saran Wrap which was in tum held in place by a wrapping of athletic adhesive tape.
In short, both methods varied only in the material initially covertng the test material with the exposure site occluded in both cases with Saran Wrap . After removal of the wrappings, the exposure site was wiped with gauze soaked with distilled water to remove residual test material.
Rats were exposed for 24 hr to a single application of test material moistened with an equivalent weight of distilled water. The test material was held in contact with the skin by wrapping the torso of the rat with Elastoplast elastic bandage lined with polyethylene.
A Saf-T-Shield collar was placed around the neck of each test rabbit during the exposure pertod to prevent the animal from removing the wrappings while allowing free access to food and water. The rats were unable to reach the wrappings in a sutficient manner to remove them and for this reason a restraining device was not used. After removal of the wrappings, the exposure site was wiped with gauze soaked with distilled water to remove residual test material .
One group of animals from each species received test material via an sc injection. The test materials were administered as a 1 % solution (w/v) in distilled water at a volume of 1 ml/kg. Injections were made dorsally on each animal. These animals had been clipped of hair at the same time and manner as those used for topical exposure.

Duration of exposure:

24 h

Doses:

- rabbit: under plastic cover dose levels of 0.5, 0.1, 0.01, and 0.001 g/kg bw and under gauze at levels of 1.0, 0.5, and 0.1 g/kg bw.
- rat: under plastic cover dose levels of 0.01, 0.1, and 0.5 g/kg bw
- s.c. injection with a dose of 0.01 g/kg bw in both species

No. of animals per sex per dose:

10 female animals per dose except 0.5 g/kg bw PHZ under gauze cover where n = 5

Control animals:

yes, concurrent vehicle

Details on study design:

Treatment and experimental design:
In order to investigate the effect of varying the exposure method on the toxicity of HS, rabbits were exposed to HS under plastic cover at dose levels of 0.5, 0.1, 0.01, and 0.001 g/kg bw and under gauze at levels of 1.0, 0.5, and 0.1 g/kg bw. Plastic covering was not used at the 1.0 g/kg bw dose level since earlier findings indicated such an exposure would be 100 % lethal to the rabbit. Exposure at the 0.01 and 0.001 g/kg bw dose levels was under plastic cover only since it was felt that the quantity of material would be so small as to be physically retained in the mesh of the gauze. Subcutaneous injection of HS at 0.01 g/kg bw was utilized as a rough indicator of complete dermal absorption of the test material.
To investigate the toxicity of HS in a second species, this material was topically applied to rats at dose levels of 0.01, 0.1, and 0.5 g/kg bw. Exposure was under plastic only since an exposure method comparison was intended. As with the rabbits, the 0.01 g/kg sc injection served as a rough indicator of dermal absorption.
In order to establish a relative toxicity for HS with a known hemolytic compound, both rabbits and rats were exposed to Phenylhydrazine hydrochloride (PHZ) at dose levels ranging from 0 .01 to 0.5 g/kg bw. Exposure methods in both species were the same as described above.

Study parameters:
All animals were observed closely at least twice each day for gross signs of toxicity.
Body weights were recorded on Days -1, 0, 1, 4, 7, 11 and 14 (Day 0 being the day of application of the test material).
Blood samples were collected from all rabbits on Study Days 2, 4, and 14 via a marginal ear vein. For rats, blood samples were collected via intraorbital puncture under light ether anesthesia from randomly selected animals (5/group) on Day 2. Blood samples were collected from the remaining 5 rats from each group on Day 4 and from all 10 rats in each group on Day 14.
Methemoglobin determinations were performed on the Day 2 blood samples using an IL-282 Co-oximeter.
Erythrocyte, leukocyte, platelet, and reticulocyte counts, as well as determinations of total hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration, were determined from Day 4 and 14 blood samples.
Platelet counts were performed using an Ultra-Flow 100 counter. Reticulocyte counts were obtained from new methylene blue-stained blood smears. The remaining parameters were measured with a HEMAC automated hematology analyzer.
Animals surviving for 14 days were terminated by Surital injection followed by exsanguination. Spleens and livers were weighed and gross appearance was recorded.

Statistics:

Statistical analysis was based on a decision-tree scheme for selecting statistical procedures as described by Gad and Weil (1984). Quantitative continuous variables were intercompared for test versus control groups by employing the following statistical tests:
Bartlett's homogenity of variance, ANOVA, and Duncan's multiple range test. In cases where heterogeneous variance was indicated or where data was suspected to be nonparametric, the Kruskal-Wallis nonparametric ANOVA and Wilcoxan rank sum test were utilized.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 100 - < 500 mg/kg bw

Remarks on result:

other: LD50 for rabbit under occlusive conditions

Sex:

female

Dose descriptor:

LD50

Effect level:

> 1 000 mg/kg bw

Remarks on result:

other: LD50 for rabbit under semiocclusive conditions

Sex:

female

Dose descriptor:

LD50

Effect level:

> 500 mg/kg bw

Remarks on result:

other: LD50 for rat under occlusive conditions

Mortality:

Rabbit:
No deaths occurred when HS was applied to the skin under gauze at a dose level of 1.0 g/kg bw. In sharp contrast, 90% of the rabbits died following exposure to 0.5 g/kg bw HS under a plastic cover while 20 % mortality occurred at a HS dose of 0.1 g/kg under similar exposure conditions. The mean time-to-death was approximately 2 days at the higher dose and 5 days at the lower dose. No deaths occurred in HS-exposed rabbits at dose levels below 0.1 g/kg bw regardless of the method of exposure.
Rat:
No mortality occurred in rats exposed to HS at any of the dose levels tested .

Clinical signs:

other: Rabbit: Gross signs of toxicity with HS included skin irritation with some necrosis at the exposure site 24 hr postapplication of the test materials. Necrosis appeared more severe when the chemicals were applied under plastic than when gauze was used . Sl

Gross pathology:

Rabbit:
At necropsy, the spleens of HS-exposed rabbits appeared enlarged and dark in color with pitting of the surface noted in some cases. These effects were most evident in those rabbits exposed under a plastic cover or via sc injection. Mean relative spleen weight was slightly greater than control values with exposure under plastic at the higher dose levels and with sc injection although the difference between groups was not statistically significant (organ weight data not presented). Livers appeared dark in color in a small number of rabbits exposed to HS at dose levels of 0.1 g/kg under plastic and 0.01 g/kg sc. Liver weights were not affected.
Rat:
Principal findings at necropsy included a high incidence of enlarged and darkened spleens in animals exposed to HS regardless of the dose received or the route of exposure. Gross effects on the liver were minimal to absent. Relative spleen weights were statistically greater than control in rats exposed to HS s.c. and with topical exposure to a dose of 0.5 g/kg. No effect on the weight of the spleen was evident at lower doses with topical exposure. The increase in spleen weight was similar in magnitude with both s.c. injection and topical exposure. Liver weight was not affected.

Other findings:

Hematology:
Rabbit:
HS exposure caused a marked elevation in methemoglobin at a dose of 0.5 g/kg under plastic while having only a slight effect on methemoglobin levels when administered under gauze at 1.0 g/kg no effect under gauze at dose levels of 0 .5 g or lower. Because of instrument malfunction, 48-hr methemoglobin values could not be determined from rabbits exposed to HS at the lower dose levels until 4 days following initial exposure. However, based on the methemoglobin levels at this time, it is reasonable to assume that methemoglobin levels were elevated in these groups at 48 hr with possibly all but the lowest dose level (0 .001 g/kg). The methemoglobin levels of rabbits exposed to HS both topically and sc were virtually similar at the 0 .01 g/kg dose level at this time .
The number of erythrocytes (also total hemoglobin and hematocrit) was significantly reduced in a dose-related manner in those rabbits exposed under plastic or via sc injection. The erythrocyte counts of animals exposed to HS under gauze were only slightly depressed with statistical significance evident only at the highest dose level (1.0 g/kg). Reticulocyte counts were elevated in a dose-related manner in rabbits exposed to HS under plastic and via sc injection with little change in reticulocyte numbers noted in animals exposed to HS under gauze . With both parameters, the effects seen at a dose level of 0.01 g/kg were nearly similar with plastic exposure and sc injection.
Heinz bodies were observed 4 days after initiation of exposure in erythrocytes of Hstreated rabbits with the incidence being markedly higher in those animals exposed under plastic or via sc injection.
White cell counts (data not presented) were statistically elevated in the groups exposed to HS under plastic or via sc injection. However, approximately 25% of the increase at higher doses resulted from circulating immature (nucleated) erythrocytes. Despite some slight effects still noted in animals exposed under plastic or via sc injection, recovery was well underway at this time.
Rat:
Methemoglobin was statistically elevated over control values in all HS exposed groups with the greatest increase occurring with topically applied HS at a dose level of 0 .5 g/kg and sc injected HS at a dose of 0.01 g/kg. The methemoglobin level of rats exposed to HS at a dose of 0 .01 g/kg was approximately three times greater with sc injection than with topical application.
A slight statistically significant reduction in erythrocyte number (p< 0.05) occurred in rats which received 0.01 g/kg HS via sc injection. Topical exposure at 0.5 g/kg also caused an apparent reduction in mean number of circulating erythrocytes although the difference was not statistically significant possibly because of a larger standard deviation. Reticulocytes were elevated in rats exposed topically to HS at dose level of 0.01 and 0.5 g/kg or with sc injection. The increase in reticulocytes at the 0.01 and 0.5 g/kg dose levels was not statistically significant. Heinz bodies were not observed in circulating erythrocytes. Platelet and leukocyte numbers were slightly elevated with topical exposure to HS at 0.5 g/kg and following sc injection.
Erythrocyte and reticulocyte numbers for rats 14 days after initial application of HS were statistically lower than control values(p < 0.01) in those animals exposed to HS via sc injection (0.01 g/kg) or topical exposure (0.5 g/kg) only . The magnitude of the effect was roughly similar for both routes of exposure despite the 50-fold difference in dose level Reticulocytes were elevated only with topical exposure at 0.5 g/kg . Reticulocyte numbets were statistically lower than controls following topical exposure to HS at 0.01 g/kg, but this was felt to represent biological variation and not be related to HS exposure.

Under plastic cover (a), HA was more toxic than under conventional gauze cover 
(b) [rabbit, 24 h exposure]:
                dose      lethality(<=2d)   Methb(mean)
----------------------------------------------------------
a)             0.01 g/kg        0            ca. 2.4 %(2d)
               0.1  g/kg       20 %          ca. 20 % (3d)
               0.5  g/kg       90 %          ca. 61 % (2d)
b)             0.1  g/kg        0 %          ca.  2 % (2d)
               0.5  g/kg        0 %          ca.  2 % (2d)
               1.0  g/kg        0 %          ca.  6 % (2d)
----------------------------------------------------------
LD50 was in the range of 70 to 200 mg base/kg. (Compare
under b): 620 < LD50 < 820 [mg base/kg] [Allied Corp.]

The more dramatic impact under a) was also reflected in the
increased number of animals with Heinz body formation down
to 0.01 g/kg. Characteristic was dermatitis with some
necrosis 24 h p.a., appearing more severe when HAS was
administered under a). Cyanosis and splenomegaly were
evident under a) at dose levels of 0.1 and 0.5 g/kg.

An acute NOAEL(dermal) of 0.01 g/kg (= ca. 4 mg base/kg)
can be derived under plastic cover (disregarding formation
of Heinz bodies as criterion for adverse effect); otherwise
NOEL (not NOAEL) = 0.001 g/kg (= ca. 0.4 mg base/kg!) under
stringent condition in rabbits (Allied Corp.).

The authors speculate that the plastic cover may maintain a
local moist environment, hydrating both the skin and the
test material while gauze could have the opposite effect by
absorbing moisture, thus reducing the water content of the
stratum corneum.

Additional information

Acute Oral Toxicity:

There are valid data available for the assessment of the acute oral toxicity of hydroxylammonium sulfate. Five male and five female Sprague-Dawley rats were treated each with 200, 400, 500, 640, 800, 1000, 1250, 1600 mg/kg bw, administered as oral application of 2 % and 4 % aqueous solutions of chemically pure test material under standardized conditions; the test method was comparable to OECD guideline 401. The animals were observed for 14 d, necropsy was performed even with the survivors. The LD50 was 642 mg/kg bw for male and female rats. Clinical signs observed included dyspnoea, trembling, convulsions, tremors and lateral position. At necropsy, blue-violet discoloration and distension of the spleen were detected.

An oral LD50 of approximately 200 mg/kg bw was detected for female cats in a study on the methaemoglobin formation properties of hydroxylammonium sulfate (purity 99.5 %). Toxic effects following oral application of single doses of 50 mg/kg bw to 1 male and 1 female cat and single doses of 200 mg/kg bw to 3 male and 3 female cats were assessed (aqueous solution dispensed by gavage).The application of 50 mg/kg was survived by both cats which demonstrated methaemoglobin concentrations of 12.1 % resp. 21.6 % at the 4-hours observation time. After application of 200 mg/kg bw hydroxylammonium sulfate 0/3 male and 2/3 female cats died; in this group between 10.0 % and 41.9 % methaemoglobin was detected 4 hours after substance application. Clinical signs in the 50 mg/kg bw group included increased salivation, cyanosis and vomiting; in the 200 mg/kg bw group repeated vomiting, increased salivation, apathia, cyanosis, mydriasis and lateral position were observed. Based on these effects, a LD50 > 50 and < 200 mg/kg bw for females and > 200 mg/kg bw for males for cats was derived. Deaths occurred 2 days after application, the surviving animals recovered within 9 days. Necropsy of the cats that died revealed weak heart muscles, acute passive hyperemia, discolored liver and lungs.

 

Acute Inhalation Toxicity:

There is no valid information available on studies performed in order to detect the inhalation LC50 of hydroxylammonium sulfate nor is there any information on methaemoglobin formation after inhalation of the substance. No studies using hydroxylammonium sulfate vapours are available. Three inhalation risk tests with rats demonstrate that inhalation of saturated vapours (saturated at 20°C) did not cause severe toxic effects in this species. However, due to the physico-chemical properties of the substance (solid at room temperature and salt character) the vapour pressure of hydroxylammonium sulfate is assumed to be very low, the concentration of hydroxylammonium sulfate after saturation in air must also be very low. Hence, it is questionable whether the method used in the two reported inhalation studies led to significant, toxicologically relevant concentrations of hydroxylammonium sulfate. Saturated vapours of chemically pure hydroxylammonium sulfatewere produced by conducting air through a 5 cm layer of the substance at 20°C. These saturated vapours were inhaled by 12 rats for a period of 8 hours. None of the rats died and no clinical signs were observed. Autopsy revealed no relevant findings.

In a second study 12/12 rats survived a 7-hour exposure period to saturated vapours ofhydroxylammonium sulfate (purity 99.5 %). Saturated vapours were produced by conducting 200 l air per hour through a 5 cm layer of the substance at 20°C (method according to Smyth et al., 1962). The resulting vapours were let into one-animal inhalation chambers for a 7-hour exposure periods and animals were observed following exposure after 3 min, 10 min, 30 min, 1 hour, 3 hours, 7 hours and thereafter daily. All animals survived within a 14-days observation period. No clinical signs were observed. At necropsy, no changes were detected.

Acute Dermal Toxicity:

There are valid data available for the assessment of the acute dermal toxicity of hydroxylammonium sulfate. The acute dermal toxicity of hydroxylammonium sulfate is different for rats and rabbits. Furthermore, the findings were different depending on the type of coverage used following substance application.

A test with rats resulted in a dermal LD50 higher than 500 mg/kg bw. Female rats were exposed for 24 hours to a single application of hydroxylammonium sulfate (purity >98 %) moistened with water. The test material was held in contact with the skin by wrapping the torso of the rat with a polyethylene bandage. One group of animals received test material via a sc injection (as a 1 % aqueous solution). Ten animals per group were exposed to doses of 500, 100 and 10 mg/kg. Subcutaneous injection of 10 mg/kg of the substance was utilized as a rough indicator of complete dermal absorption of the test material. All animals were observed closely at least twice each day for gross signs of toxicity, blood samples were collected from all animals, methaemoglobin determination was performed on day 2. Erythrocyte, leukocyte, platelet, and reticulocyte counts, as well as determinations of total haemoglobin, hematocrit, mean corpuscular haemoglobin concentration, were determined from days 4 and 14 blood samples. No mortality occurred within this test, skin irritation of moderate incidence, and to a lesser extent necrosis and sloughing, were evident. A large percentage of the rats exposed became pale following exposure (all dose levels). This effect was evident within 24 hours and persisted to approximately 6 days, cyanosis was not observed. Other gross signs of toxicity included staining of the nares, mouth, and fore paws with brown material, yellow staining of the anogenital area, and lacrimation. Blood methaemoglobin levels determined 48 hours following initial exposure were statistically elevated over control values in all exposed groups with the greatest increase occurring in the topical 500 mg/kg group (4.0 %) and in the 10 mg/kg sc injected group (6.3 %). Heinz bodies were not observed in circulating erythrocytes. Principal findings at necropsy included a high incidence of enlarged and darkened spleens regardless of the dose level or route of exposure. Gross effects on the liver were minimal to absent.

A dermal LD50 between 100 mg/kg bw and 500 mg/kg bw was detected for rabbits in a study comparing occlusive and semi-occlusive dermal exposure. Hydroxylammonium sulfate (purity >98 %) proved strikingly more toxic when administered under occlusive dressing compared to semiocclusive dressing despite the fact that both methods included occlusion. Female Albino rabbits were exposed for 24 hours to a single topical application of test material moistened with water. The test material was covered with either a porous gauze patch or a plastic cover. One group of animals received test material via a sc injection (as a 1 % aqueous solution). Ten animals per group were exposed to doses of 500, 100, 10 and 1 mg/kg under plastic cover and doses of 1000, 500 and 100 mg/kg using gauze. Plastic covering was not used at the 1000 mg/kg level since earlier findings indicated such an exposure would be 100% lethal to the rabbit. Subcutaneous injection of 10 mg/kg of the substance was utilized as a rough indicator of complete dermal absorption of the test material. All animals were observed closely at least twice each day for gross signs of toxicity, blood samples were collected from all animals, methaemoglobin determination was performed on day 2. Erythrocyte, leukocyte, platelet, and reticulocyte counts, as well as determinations of total haemoglobin, hematocrit, mean corpuscular haemoglobin concentration, were determined from days 4 and 14 blood samples. Animals surviving for 14 days were necropsied. After occlusive skin contact with 100 mg/kghydroxylammonium sulfate 2/10 rabbits died demonstrating 18.7% methaemoglobin, in surviving animals 80 % Heinz bodies in erythrocytes were detected 4 days after exposure. After semi-occlusive skin contact with 100 mg/kg bw hydroxylammonium sulfate no deaths were noted (methaemoglobin 1.9%). After occlusive skin contact with 500 mg/kg bw 9/10 rabbits died demonstrating 60.8 % methaemoglobin concentration and formation of Heinz bodies in all animals (no quantitative data). Therefore, the LD50 in rabbits is >100 and < 500 mg/kg bw. After semi-occlusive skin contact with 500 mg/kghydroxylammonium sulfateno animals died (methaemoglobin 1.9%). Even in the 10 mg/kg bw group with occlusive exposure (none of the animals died, no methaemoglobin formation), Heinz bodies were detected in one animal 4 days after exposure. In addition, erythrocytes and reticulocyte counts were significantly decreased and increased, respectively. In contrary, within the semi-occlusive exposure groups even 1000 mg/kg bw was survived by all animals (percentage of methaemoglobin 6.2% and formation of Heinz bodies in three animals). Here, the LD50 was > 1000 mg/kg bw. The reference group with sc injection of 10 mg/kg (no mortality) revealed 5.1% methaemoglobin, and Heinz bodies were found in erythrocytes of all animals (4 days after treatment, no quantitative data). However, it is to be mentioned that there were some methodological insufficiencies concerning methaemoglobin determination on rabbits in this study.

Another test employing rabbits demonstrates again that semi-occlusive skin exposure is much less toxic. In a limit-test with rabbits the skin of 5 male and 5 female rabbits was exposed occlusively to a dose of 400 mg/kg bw hydroxylammonium sulfate (purity 99.5%) using a 50% aqueous substance solution (exposure period 24 hours). None of the rabbits died within an 8-days observation period, neither clinical signs nor local effects were detected, necropsy revealed no changes.

A dermal LD50 higher than 1500 mg/kg bw but less than 2000 mg/kg bw resulted for hydroxylammonium sulfate (no data on purity) in a further study with rabbits: Male and female rabbits from 4 test groups were treated with single applications of hydroxylammonium sulfate at doses of 2000 mg/kg bw (6 males and 4 females), 1500 mg/kg bw (5 males and 5 females), 1000 mg/kg bw (5 males) and 500 mg/kg bw (5 males and 5 females) and observed for 14 days. Control rabbits were treated with water (5 males and 5 females). The test material was placed on gauze strip and then applied to the hair free skin of the back. Seven of 10 rabbits died or were sacrificed in extremis at the high dose level after 3 days, all remaining rabbits survived. An acute hemorrhagic dermatitis was present in each of the 7 rabbits that died. Necropsy examination of the tissues revealed severe hemorrhagic necrosis of the skin characterized by massive subepithelial and dermal lesions which often extended from just beneath the epithelium to the cutaneous muscle layer, blood appeared brown. The skin lesions of the surviving animals were characterized by hemorrhage, edema, bulla formation, vascular congestion, and massive heterophil infiltrates. Changes in other tissues were not definitive of compound induced toxicity. It appeared, however, that the deaths may have been related to circulatory collapse (shock) brought on by neurogenic pain reflexes associated with the skin lesion. Lesions in the liver and kidneys supported this contention. Few compound associated changes were present in survivors. The skin contained residual but healing changes. The spleens of 3/3 high and 4/10 mid dose terminated rabbits contained increased amounts of hemosiderin pigment. Red cell damage was detected, other tissue alterations were judged to have been unrelated to hydroxylammonium sulfate treatment.

Justification for classification or non-classification

Based on the data available and in accordance with Regulation (EC) 1272/2008, classification as acute toxicant Cat. 4 following oral and dermal exposure (H302, H312) is warranted. This is in line with the current classification according to 1272/2008/EC Annex VI.