Bis(hydroxylammonium) sulphate

Acute Toxicity: dermal

Administrative data

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.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Toxicity of Hydroxylamine Sulfate following Dermal Exposure: Variability with Exposure Method and Species

Author:

Derelanko MJ, Gad SC, Gavigan FA, Babich PC, Rinehart WE

Year:

1987

Bibliographic source:

Fund. Appl. Toxicol., 8, 583-594

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

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

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): Hydroxylamine Sulfate (HS)
- Analytical purity: 98 %
- Other: test material was obtained from the Chemical Sector of Allied Corp.

Test animals

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

Administration / exposure

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.

Results and discussion

Effect levels

Effect levels 1

Sex:

female

Dose descriptor:

LD50

Effect level:

> 100 - < 500 mg/kg bw

Remarks on result:

other: LD50 for rabbit under occlusive conditions

Effect levels 2

Sex:

female

Dose descriptor:

LD50

Effect level:

> 1 000 mg/kg bw

Remarks on result:

other: LD50 for rabbit under semiocclusive conditions

Effect levels 3

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.

Any other information on results incl. tables

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.