N-isopropylhydroxylamine

Endpoint summary

Description of key information

The NOAEL for N-isopropylhydroxylamine (IPHA) in a 90-day oral gavage study was considered to be 20 mg/kg, based on decreased body weight and signs of anaemia observed at 100 and 500 mg/kg bw. These effects were considered to be treatment-related.

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:

key study

Study period:

May-November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name: IPHA I-15 Hydroxylamine (IPHA)
- Chemical name: Isopropyl hydroxylamine
- Molecular formula: C3H9NO
- CAS number: 5080-22-8
- Batch number: D609G1EGQ9
- Appearance: Colourless liquid
- Purity : 15% IPHA aqueous solution
- Manufacturer: ANGUS Chemical Company
- Manufacture date: 14 January 2016
- Expiry date: 14 January 2017
- pH: 10.6


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Room temperature, under inert gas (15-25oC, below 70 RH%)
- Safety Precautions: Routine safety precautions (lab coat, gloves, safety glasses and face mask) for unknown materials were applied to assure personnel health and safety.
Dangerous to the environment.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item will be formulated in distilled water at the appropriate concentrations according to the dose levels selected. During the dose formulation the pH of each concentration will be adjusted to below pH 10, the added volume of 10% HCl solution used for adjustment and the final pH value of the formulations will be documented in the raw data and reported.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species and strain: Crl:WI rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species and strain: Crl:WI Wistar rats
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633, Sulzfeld
- Hygienic level: SPF at the supplier, standard laboratory conditions during the study
- Justification of species/strain: The rat is regarded as suitable species for toxicology studies. Wistar rat as a rodent is one of the standard strains for repeat-dose toxicity studies.
- Number of animals: 40 males + 40 females. 5 males + 5 females for replacement purpose (no replacement was performed)
- Age of animals: young adult rats, 8 weeks old at start of treatment
- Target body weight: not exceeded ± 20% of the mean weight for each sex at onset of treatment. Males: 281 – 333 g. Females: 182 – 213 g
- Acclimation period: 7-8 days

DETAILS OF FOOD AND WATER QUALITY:
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum, and tap water from municipal supply, as for human consumption from a 500 ml bottles, ad libitum. Analytical certificate(s) for the batch(es) used, were archived with the study raw data. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary). The quality control results were archived with the study raw data. The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
Cage type: Type II polypropylene
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany suitable for the purposes of the study. Details of bedding quality were archived with the study raw data.
Light: Artificial lighting 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 21.0 – 26.8°C
Relative humidity: 31 – 78 %
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: rodents were housed up to 3 animals of the same sex and group/cage. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities.

The temperature and relative humidity were measured continuously and recorded twice daily during the study and acclimation period.

IN-LIFE DATES: From 10/11 May 2016 to 9 August 2016

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is a possible route of exposure to the test Item in humans.

Vehicle:

water

Details on oral exposure:

Ten males and ten females/group were treated daily by oral gavage, using a bulb tipped gastric feeding tube attached to a syringe. A constant dose volume (10 mL/kg bw) was administered to all animals. Control animals were treated concurrently with the vehicle only. The actual volume administered was calculated and adjusted based on each animal’s most recent body weight. Control animals were treated concurrently with the vehicle only.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item has been be formulated in distilled water at the appropriate concentrations according to the dose levels selected. During the dose formulation the pH of each concentration has been adjusted to below pH 10, the added volume of 10% HCl solution used for adjustment and the final pH value of the formulations documented in the raw data and reported. Formulations were prepared for a maximum period of use of 7 days. To verify the concentration of the test item in formulations, representative samples were taken and analysed from each concentration at least four times during the study.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Dose / conc.:

20 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
Tthe doses were selected based on available information and results of 7-day study by oral gavage in rats performed at CiToxLAB – Hungary Ltd. (Study code: 16/004-101PE). Based on these results, the 500 mg/kg bw/day dose was considered to be acceptable as top dose in the 90-day repeated dose study. The oral route was selected as it is a possible route of exposure to the test Item in humans.

During the acclimation period, the animals were assigned to their respective dose groups by randomization based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; the software PROVANTIS v.9 was used in order to verify homogeneity/variation among/within groups. Males and females were randomized separately.

Positive control:

not applicable

Observations and examinations performed and frequency:

MORTALITY AND CLINICAL OBSERVATIONS
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each working day). The animals which show clinical signs considered severe were isolated. General clinical observations were made once a day. Detailed clinical observations were made on all animals outside the home cage in a standard arena prior to the first treatment (to allow for within-subject comparisons) and weekly thereafter, in the morning hours (am).

NEUROLOGICAL ASSESSMENT (Functional Observation Battery)
Towards the end of the treatment period on Day 78-83, each animal was subjected to the functional observation battery, including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. A detailed assessment for neurotoxicity effects were made on the basis of these measurements (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).

Parameters such as body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated.

To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured.

Fore/hind grip strength measurements was conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs are allowed to grip the support bar and pulled back until they release the bar; the device measures the maximum grip strength. This was performed 3 times for each animal on the test day. The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data.

Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1 hour observation time, when DVD recording of movement was made. Recording was made for a duration of 30 min, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Data from the high dose and control group were evaluated for distance travelled in 5 minute segments. The data from the 5 minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.

BODY WEIGHT
Body weight was recorded with a precision of 1 g at randomisation (pre-treatment period), on the first day of treatment (Day 1, prior to start of treatment), then weekly, including on Day 90 (last treatment day) and prior to necropsy (if scheduled necropsy, fasted, on Day 91; or at death, for animals found dead during the course of the study). Weekly body weight and body weight gain were reported.

FOOD CONSUMPTION
The determination of food consumption was performed for all groups once a week. The remaining, non-consumed food were weighed weekly from Day 8 with a precision of 1 g. Weekly food consumption were calculated for reporting purposes.

OPTHALMOLOGY EVALUATION
Ophthalmoscopic examination was conducted in all animals before treatment, on Day -1, and in the Control (Group 1) and High dose (Group 4) animals, on Day 87.

EXAMINATION OF VAGINAL SMEARS
Prior to necropsy, the oestrus cycle of all females were determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution. The smear were examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

Sacrifice and pathology:

TERMINAL PROCEDURES
Unscheduled deaths: Gross pathology was performed on every animal irrespective of the date of its death: animal found dead or euthanized pre-terminally during the study for humane reasons. Dead animals were necropsied as early as possible after discovery, according to CiToxLAB Hungary Ltd. SOPs.

Scheduled Euthanasia: Necropsy and macroscopic examination were performed on all surviving animals, at the end of treatment period, on Day 91 (after the sample collection for clinical pathology evaluation). The animals were euthanized by exsanguination under pentobarbital anaesthesia.

MACROSCOPIC EVALUATION
After exsanguination the external appearance was examined, all orifices, and the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. In addition, bone marrow smears from the femur of each animal were prepared at necropsy (but not examined).

ORGAN WEIGHT
The following organs were trimmed of fat and weighed in surviving animals:
Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Prostate, Seminal vesicles with coagulating glandsp, Spleen, Testes, Thymus, Thyroids with parathyroids, Uterus including cervix.
Paired organs were weighed together. Absolute organ weights were measured, and relative organ weights to the body and brain weights were calculated and reported.  

HISTOPATHOLOGY
On completion of the macroscopic examination the following tissues and organs were retained from all animals:
Gross findings, Lungs with bronchi Skeletal muscle (quadriceps)
Adrenals Lymph node Small intestine
Animal identification Ovary Spinal cord
Aorta Oviduct Spleen
Brain Pancreas Sternum with marrow
Epididymis Pituitary Stomach
Eye with the optic nerve Prostate Testis
Oesophagus Salivary gland (including
mandibular, sublingual
and parotid glands) Thymus
Femur with marrow Thyroid with parathyroid gland
Heart Tongue
Kidney Sciatic nerve Trachea
Large intestine Seminal vesicle with
coagulating gland Urinary bladder
Extraorbital lachrymal gland Uterus
Harderian gland Skin, subcutis with
mammary gland (inguinal) Vagina
Liver

The eyes with the optic nerve and the testes with epididymides were preserved in modified Davidson’s fixative; all other organs in 10% buffered formalin solution.

The retained tissues and organs for histological examination were embedded in paraffin wax, sections were cut at 4-6µ by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope. Full histopathology was performed in Groups 1 (Control) and 4 (High dose), any macroscopic abnormalities from other groups and in animals found dead during the study. In addition, as Test Item related microscopic findings were identified in the spleen, kidney and liver of High dose animals, these organs from the Low and Mid dose were processed to slides and examined microscopically.

Other examinations:

At the end of the treatment period, prior to scheduled necropsy on Day 91, clinical pathology investigations (haematology, coagulation, clinical biochemistry and urinalysis) were conducted in all surviving animals.

After an overnight period of food deprivation of animals, 3 blood samples were collected by heart puncture under pentobarbital anaesthesia, for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

Urine collection was conducted over approximately 16 hours, during an overnight period of food deprivation of animals, which were placed in metabolic cages. The evaluation of the urine samples was performed by observation (e.g. colour, appearance) or test strips as applicable.

Statistics:

Data were collected using the software PROVANTIS v.9 or recorded on data Data were collected using the software PROVANTIS v.9 or were recorded on the appropriate forms as per relevant SOPs, then tabulated using PROVANTIS v.9, Microsoft Office Word and/or Excel, as appropriate. Group mean and standard deviation were calculated for numerical data. Statistical analysis was performed using SAS 9.2 (built in Provantis System)
 
The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data: the normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests show no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis was the better option when the normality and heterogeneity assumptions implicit in the tests are adequate. If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In control, one male rat (1002) had red discharge from Day 7 until the terminal euthanasia. This was described as incidental or common background. In the low dose group all animals were clinically normal during the whole treatment period.

In mid dose 1 male (3003) had a subcutaneous nodule recorded from Day 3 (<1 cm, diameter approx. 1.5 cm), this was continuously growing (terminal size was 8-10 cm), from Day 56 the animal’s front legs were inhibited from movement because of the nodule until then end of the study. Red discharge from the nose from Day 43-49 and Day 54-90 and 1-2 cm scar was present Day 71-90. All other animals were clinically normal during the study. Findings of animal 3003 were described as not test item related and incidental.

In high dose there were 1 male and 1 female found dead (Day 83 and 82). Three males were described with red discharge at the nasal area and 1 female (4504) also with red discharge at the nasal area and coloured discharge at the chin during the last 2 weeks of the study. These pre-mortality clinical signs were considered test item related, no other clinical signs in the study were considered to be treatment related

Mortality:

mortality observed, treatment-related

Description (incidence):

One high-dose male was found dead on day 83. Discharge red of the eyes from day 81-82 preceded the death, there were no other clinical signs observed before. One high-dose female was found dead on day 82. Discharge red from the vagina preceded the death and there was a palpable swelling under the skin in the abdominal region from Day 80-82, there were no other clinical signs observed before.

For both animals at necropsy the following observation were recorded: dark discoloration in all lobes of the lungs, but the lungs were not collapsed, discoloration of the perinasal area of the fur dry material with dry, red material. No clear cause of death was established for these animals; however deaths were attributed to the test item. Treatment-related moderate increased hematopoietic cells (erythroid) and slight increased pigmented (yellow) macrophages in the spleen were noted in the male rat. No treatment-related effects were seen in the female rat.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were test item related effects observed on the animal body weights or body weight gain values during the study. Statistically significantly lower mean body weights were recorded in high dose males from the second week of the treatment period until the terminal euthanasia. On Day 90 the difference was -14.8% compared to the control. In high dose females it was lower during the treatment period from Day 8 without statistical significance, except on Day 83 (-8.5% lower). The overall body weight gain values were statistically significantly lower that of the control in both sexes, for males -30.2% and -25.3% for females. The body weight gain was statistically significantly lower in males during most of the second half of the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were test item related effects on animal food consumption ascribed to IPHA I-15 Hydroxylamine (IPHA) administration under the conditions of this study. For both sexes in the high dose, the food consumption was statistically significantly lower than the control throughout most of the study. The overall food consumption was, -17.0% for males and -14.4% lower for females. In the mid dose the food consumption was slightly lower in both sexes with occasional statistical differences.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test item related changes compared to pre-treatment or/and the control were noted at ophthalmoscopy examination. All examined animals were found to be normal.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology parameters evaluated at the completion of the 90-day treatment period showed adverse effects or findings that were test item related. In high dose animals in both sexes the results indicated test item related anaemia, with statistically significantly lower RBC, HGB, Hct percentage, MCV, MCH. Also the Reticulocytes percentage was increased significantly in mid and high dose in both sexes confirming a compensated anaemia in the high and mid dose groups. WBC was statistically significantly lower in mid and high dose females only, not in males. The mean values were well within the historical control range for this parameter. The relationship between the statistical differences and treatment is equivocal; there were no other changes in these animals to suggest a toxicological effect on this parameter. Also, the high dose males only had a statistically higher PPT, however, the values were all well within the historical range. Furthermore, there was no change in females. The statistical difference in PPT is not attributed to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clearly adverse test item related effects on the clinical chemistry parameters evaluated at the completion of the treatment period. The high dose animals were clearly affected in clinical chemistry values, particularly the electrolytes. Bilirubin was increased in High dose males, related to the anaemia effect seen above. Bile acid showed statistically significantly higher values in mid and high dose males and in High dose females. This parameter has high standard deviations and the biological significance of the statistical differences are not clearly related to treatment. Statistical differences in Albumin, protein and urea levels in high dose males may be related to histopathological changes seen in the liver of this group. The statistical difference recorded in high dose females was well within the historical range for this parameter and is not ascribed to treatment.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no clearly adverse test item related effects on the urinary parameters evaluated at the completion of the treatment period. The urinary pH was statistically significantly higher than control in the high dose females , in males it was comparable to the control. The pH in the high dose females was well within the normal historical range, so the difference is not clearly related to treatment. There were no other significant differences in urinary parameters between groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no findings in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups.

In high dose males during the assessment of grip strength statistically significantly lower values were noted in the average fore and hind limb. This difference may be attributed to lower bodyweight at the High dose level (animals are less well developed than controls).

In the landing splay-test in high dose males and and females there were lower values compared to the control mean, however since the animals were smaller, a lower value is expected; this is not considered to be a direct adverse effect of treatment. In mid dose females there was a statistical difference (p<0.05) however the values were all well within the historical range, this difference is not attributed to treatment.

All dose groups of males and females had a normal locomotor activity; in all cases, the initial activity was high, with reduces activity in each 5 minute period to an approximate plateau by about 20-30 minutes. There were no statistical significance between the high dose animals and the control when evaluating the overall distance travelled (0-60 min, cm). Sporadic statistical differences at individual 5 minute periods were considered not related to treatment. The test item did not increase or decrease normal locomotor activity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute and relative (to body) spleen weights were statistically significantly higher (p<0.01) in both sexes at the mid and high dose levels. These changes in spleen weights corresponded with compensated treatment-related regenerated anaemia (↓RBC, ↓HGB, ↓Hct, ↓MCHC, ↓MCV, ↑Retic%) noted in both sexes at mid and high dose levels. The relative (to body and brain) kidney weights were statistically significantly higher (p<0.01) in high dose male and female groups. The male liver weights were statistically significantly higher only when adjusted to body in mid dose and high dose males. In the females, liver weights were statistically significantly higher at high dose level. In mid dose females, liver weights appeared to be higher, but statistical significance was not reached. Other statistically significant differences in organ weights were considered as secondary changes due to the body weight effects, not a direct effect of the test item, or as normal variations, within the expected control range.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Dark/red discoloration of the collapsed lungs and dry red material at the perinasal fur considered to be either incidental or agonal/post mortem, were observed in both found-dead animals at necropsy. At scheduled necropsy, treatment-related macroscopic findings were seen in the spleen (100 and 500 mg/kg bw/day) and kidney (500 mg/kg bw/day). Enlargement of the spleen was recorded in 1/10 Mid Dose and 2/9 High Dose males. Enlargement of the kidneys was observed in 1/9 High Dose males. These gross changes were in correlation with treatment-related findings seen by light microscopy. Other changes were incidental or background.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in the spleen, kidney and liver in animals dosed at 500 mg/kg bw/day and microscopically examined.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Treatment-related moderate increased haematopoietic cells (erythroid) and slight increased pigmented (yellow) macrophages in the spleen were noted in the found-dead male. No treatment-related effects were seen in the found-dead female. Congestion and/or haemorrhage of the lungs and inflammation of the prostate were regarded as agonal and incidental, respectively.

SPLEEN
Aat 20 mg/kg bw/day, no treatment-related changes were recorded. At 100 mg/kg bw/day, minimal to mild increased hematopoietic cells (erythtroid) in 9/10 males and minimal to mild increased numbers of pigmented (yellow) macrophages in 8/10 males, were detected. In the females, minimal increased hematopoietic cells (erythtroid) in 1/10 animal and mild increased numbers of pigmented (yellow) macrophages in 2/10 rats, were seen. In high dose (500 mg/kg bw/day) minimal to moderate increased hematopoietic cells and minimal to slight increased numbers of pigmented macrophages in 9/9 males were present. Both changes had mostly slight severity. In the females, minimal to slight increased hematopoietic cells occurred in 3/9 splenic sections. These changes correlated with statistically significantly higher absolute and relative (to body and brain).

KIDNEY
At 20 and 100 mg/kg bw/day, no treatment-related changes were present. At 500 mg/kg bw/day, minimal multifocal accumulation of pigment (yellow) within the cytoplasm of cortical tubules in 4/9 males and 1/9 female was noted. In addition, minimal or mild focal/multifocal tubule degeneration (cortex) in 4/9 males was seen. There was no evidence of tubule degeneration in examined female’s kidneys. These alterations corresponded with statistically significantly higher relative (to body and brain) kidney weights. Under the conditions of this study, accumulated pigments were regarded as non-adverse. In addition, minimal or slight tubule degeneration (cortex) affected kidneys of 4/9 males. Tubule degeneration was considered as an adverse change. No tubule degeneration was present in examined female’s kidneys.

LIVER
At 20 and 100 mg/kg bw/day, no treatment-related changes were present. At 500 mg/kg bw/day, centrilobular hepatocellular vacuolation (microvesicular) altered liver was seen. Hepatocytes partially or completely filled by numerous small vacuoles had a “foamy” appearance. Minimal to moderate (predominantly slight) intensity was described in 8/9 male rats. No hepatocellular vacuolation was detected in the females. When taken in considerations statistically significant changes in liver biomarkes suggested hepatic dysfunction such as ↓total proteins and↓albumin in high dose males, this vacuolation was considered to be adverse under the conditions of this study. Other changes were incidental or background. Additionally, there was one mid dose male (3003) histopathologically examined due to subcutaneous mass recorded at the cervical area. This male had enlarged spleen and small thymus. Spontaneous adenocarcinoma of mammary gland, spontaneous lymphoma in the spleen and liver, moderate decreased cortical lymphocytes of the thymus were observed for this male by light microscopy.

Other effects:

no effects observed

Description (incidence and severity):

There were no adverse or test item related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

kidney

liver

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Under the conditions of this study, test item related effects were observed after 90 days of treatment in the high dose group; effects included death, reduced body weight gain and, signs of anaemia. Spleen weight and histopathology confirmed the compensated anaemia effect in high and mid dose animals. Liver weights were increased in the high dose group of both sexes, with a similar trend in mid dose males, histopathology showed centrilobular hepatocellular vacuolation in high dose males only. Kidney weights were higher in high dose males and females, with some tubule degeneration in high dose males only. In the high dose group, there were clear signs of adverse effects. In the mid dose group the changes were minimal, but there was some evidence for a fully compensated anaemia. There were no adverse effects of treatment in the low dose group of either sex. The NOAEL (no observed adverse effect level) was considered to be 20 mg/kg bw/day.

Executive summary:

IPHA I-15 Hydroxylamine (IPHA) was administered by daily oral gavage to Crl: (WI) rats dosed at 20, 100 and 500 mg/kg bw/day for 90 days. Two animals died during the study in the high dose (one male on Day 83, one female on Day 82). No clear cause of deaths was established for these animals but deaths were attributed to the test tem. Test item related clinical signs were observed during the study at the 500 mg/kg bw/day dose level, such as red nasal discharge and coloured discharge on the chin, during the last 2 weeks of the study. There were no findings in the animal behaviour, general physical condition, in the reactions to different type of stimuli in the control or treated groups. The overall body weight gain values in high dose were statistically significantly lower than that of the control in both sexes, for males -30.2% and -25.3% for females which was considered to be test item related.. There were test item related effects on animal food consumption. The overall food consumption was -17.0% lower for males and -14.4% lower for females in the high dose. Haematology parameters showed adverse effects or findings that were test item related. In high dose animals test item related anaemia was seen, with statistically significantly lower RBC, HGB, Hct percentage, MCV, MCH. Also the reticulocytes percentage was increased significantly in mid and high dose in both sexes confirming a compensated anaemia. There were no adverse effects of anaemia seen in the low dose group of either sex. There were no clearly adverse test item related effects on the clinical chemistry parameters. There were no clearly adverse test item related effects on the urinary parameters evaluated at the completion of the treatment period. Changes in spleen weights in high dose males and females corresponded with compensated treatment-related regenerated anaemia (↓RBC, ↓HGB, ↓Hct, ↓MCHC, ↓MCV, ↑Retic%). The relative (to body and brain) kidney weights were statistically significantly higher (p<0.01) in high dose male and female groups. These changes probably reflect treatment-related microscopic findings. The male liver weights were higher when adjusted to body in mid dose and high dose males. In the females, liver weights were higher at high dose level. Enlargement of the spleen was recorded in 1/10 mid dose and 2/9 high dose males. Enlargement of the kidneys was observed in 1/9 high dose male.

In conclusion, under the conditions of this study, test item related effects were observed after 90 days of treatment in the high dose group; effects included death, reduced body weight gain and, signs of anaemia. Spleen weight and histopathology confirmed the compensated anaemia effect in high and mid dose animals. Liver weights were increased in the high dose group of both sexes, with a similar trend in mid dose males, histopathology showed centrilobular hepatocellular vacuolation in high dose males only. Kidney weights were higher in high dose males and females, with some tubule degeneration in high dose males only. There were no adverse effects of treatment in the low dose group of either sex. The NOAEL (no observed adverse effect level) was considered to be 20 mg/kg bw/day.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

21 March - 20 April 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was not conducted according to guidelines but was conducted according to GLPs and the report contains sufficient data for interpretation of study results

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal husbandry conformed to standards set forth in "Guide for the Care and Use of Laboratory Animals", DHEW Publication No. (NIH) 85-23.

Thirty-six male and 36 female CD rats were received in good condition from Charles River Breeding Laboratories, Inc., Kingston, New York on February 27. 1990. Upon receipt, the animals were 22 days old. Each animal was observed by a qualified technician and weighed. The animals were housed individually in stainless-steel wire-mesh cages suspended above cage paper. During the acclimation period, each rat was observed twice daily for any changes in general appearance or behavior. Animals considered suitable for study were housed for a 21-day acclimation and pretest period.

The basal ration, Agway@ProLab RHH 3200 Heal (Agway, Inc., Syracuse, NY) and tap water were provided ad libitum. The basal diet Is a certified feed with appropriate analyses provided by the manufacturer. Tap water supplying the facility is monitored for contaminants at periodic intervals according to FDRL Standard Operating Procedures.

The rats were assigned sequential temporary numbers (Waverly Number). This identification was utilized until the rats were randomized into treatment groups. Pretest data were collected the week of March 12, 1990.

Animal selection for assignment to study and treatment groups was based on pretreatment physical examinations and body weights. These data were reviewed by the study director prior to animal selection. All rats that displayed abnormalities were eliminated from consideration for study use.

Rats judged to be suitable for testing were assigned randomly to groups by stratified randomization according to body weight using a computer
generated random number sequence. These animals were arranged into study groups and assigned permanent identification numbers and ear notched.

Route of administration:

oral: gavage

Vehicle:

other: distilled water

Details on oral exposure:

The test solutions were administered orally by gastric intubation once daily for at least 28 consecutive days. A dose volume of 10.87 ml/kg was used for all dose levels. The appropriate amount of test article was mixed with distilled water. The control animals received distilled water only at a volume of 10.87 ml/kg. All doses were adjusted to 100% active, based on the test article being 18.4% active.

Approximately 60 ml of dosing solution was dispensed into 7 bottles for each dose level. The head space of each bottle was topped with nitrogen
and placed into a refrigerator. Solutions were brought to room temperature before administration to the animals. Dosing solutions were prepared fresh weekly.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing solutions were analyzed for concentration and stability for study days 0, 7, 14, 21 and 28.

All dosing solutions were analyzed on a Varian 3700 Gas Chromatograph (GC)
Column: 6 ft. x 2 mm id, 5% Carbowax 20M on Chromosorb 103 glass column
Detecter: Flame Ionization
Detector Temperature: 250°C
Injector Temperature: 220°C
Initial Column Temperature: 140°C
Temperature Ramp: 1O°C/minute
Final Temperature: 200°C
Initial Temperature Hold: 6 minutes
Final Temperature Hold: 2 minutes
Carrier Gas: Helium
Carrier Rate: 25 ml/minute
Injection Volume: 2 ul and 5 ul

Standard Preparation
Three standard solutions were prepared by diluting known quantities of NIPHA with distilled water. Concentrations ranged from approximately 0.3 mg/g to 19.0 mg/g. Injections of 2 ul or 5 ul were made into the GC to obtain peak areas for a standard curve.

Percent of theoretical concentrations ranged from 93.9-115.8%. Test material was shown to be stable for at least 7 days (at the lowest dose, the test material was shown to be stable in water for 20 days).

Duration of treatment / exposure:

Daily for 28 days

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 20, 500, and 1000 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5 males and 5 females/dose level

Control animals:

yes, concurrent vehicle

Details on study design:

This study was designed to evaluate the potential subchronic oral toxicity of the test article, N-isopropylhydroxylamine, NIPHA, when administered to Sprague-Dawley rats for at least days. NIPHA was administered by gavage once daily to three groups of animals each composed of five male and five female rats, for at least 28 days. Dosage levels of 20, 500 and 1000 mg/kg/day were chosen for the study and were administered at a dosage volume of 10.87 ml/kg in distilled water. For comparative purposes, a concurrent vehicle control group, also composed of five male and five female rats, received distilled water on a comparable regimen at 10.87 ml/kg. The animals were observed once in the morning, one hour after dose administration and once in the afternoon for signs of overt toxicity. Detailed physical examinations, individual body weights and food consumption were recorded
weekly. Clinical laboratory studies were conducted on all animals at study termination. Complete necropsy examinations were performed on all rats that were sacrificed after 28 days of dosing and organ weights obtained. Microscopic examinations were conducted on selected tissues collected for
animals in the control and high dose groups.

Positive control:

No data.

Observations and examinations performed and frequency:

Body Weights:
Individual body weights were recorded 7 days prior to study initiation, on the first day of test material administration and weekly thereafter.

Food Consumption:
Individual food consumption was measured 7 days prior to study initiation, on the first day of test material administration and weekly thereafter.

Physical Examinations:
All animals received a detailed physical examination prior to study initiation and weekly thereafter.

Post Dose Observations:
All animals were observed for pharmacotoxic signs approximately one hour after dose administration.

Mortality Checks:
All animals were observed for mortality and overt signs of toxicity twice daily at least 6 hours apart.

Sacrifice and pathology:

Clinical Laboratory Studies
The following clinical laboratory studies were conducted on 5 male and female rats prior to study initiation. These animals were sacrificed and discarded. The same determinations were made on all animals after 28 days of dosing. Blood was drawn from the periorbital plexus. The animals were under light ether anesthesia during blood collection. The animals The animals were fasted prior to blood collection and urine was collected during the fasting period.

Hematology:
Hemoglobin
Hematocrit
Erythrocyte count
Total leukocyte count
Differential leukocyte count
MCV
MCH
MCHC
Platelet count

Clinical Chemistry:
Urea nitrogen
Aspartate aminotransferase
Alanine aminotransferase
Glucose (fasting)
Creatinine
Bilirubin. total
Phosphorus
Protein, total
Albumin
Globulin
Sodium
Potassium
Chloride

Urine Analvsis:
Appearance
Specific gravity
Occult blood
Protein
pH
Microscopic examination of formed elements
Bilirubin
Urobilinogen
Ketones
Glucose

Pathology
Necropsy: A complete necropsy examination was conducted on all rats
that were sacrificed after 28 days of dosing. Necropsies were performed under
the supervision of a board certified veterinary pathologist. Animals were
COz surfaces and orifices, the cranial cavity, carcass, the external and cut
surfaces of the brain, the thoracic, abdominal and pelvic cavities and their
viscera and the cervical tissues and organs. The following organs and tissues
10% 2)
Bone with marrow (sternebrae)
Brain (forebrain, midbrain,
2)
I leum
Co 1 on
Rec tum
Kidneys (2)
Liver (sections of two lobes)
tun@ (2)
Lymph node (mesenteric)
2 )
mandibular
2) 1
medial is)
cervical,
Sp 1 een
2) - Cboth 2)l
- 5 -
90.3873.013/Page 11 of 202
DOW CONFIDENTIAL - Do not share without permission
Urine Analvsis:
Appearance
Specific gravity
Occult blood
Protein
pH
FDRL Study No. 90.3873.013
Bi \ irubin
Urobilinogen
Ketones
Glucose
Microscopic examination of formed elements
Pathology
Necropsy: A complete necropsy examination was conducted on all rats that were sacrificed after 28 days of dosing. Necropsies were performed under the supervision of a board certified veterinary pathologist. Animals were euthanized using CO2 gas. Gross necropsy included examination of the external surfaces and orifices, the cranial cavity, carcass, the external and cut surfaces of the brain, the thoracic, abdominal and pelvic cavities and their viscera and the cervical tissues and organs. Approximately 40 organs and tissues were removed from each animal and fixed in 10% neutral buffered formalin.

Lungs were inflated with 4% formaldehyde-1% glutaraldehyde in 176 mOsM phosphate buffer via the airways. Urinary bladders were distended with 10% neutral buffered formalin and left unopened for examination following fixation. Eyes were fixed in Zenker's solution.

Organ Weights: The following organs were weighed.
Adrenals
Brain
Heart
Kidneys
Liver
Ovaries
Testes
Relative organ weights (organ/body weight and organ/brain weight ratios) were calculated.

Microscopic examination of paraffin embedded hematoxylin and eosin stained tissue sections were performed on the following tissues for all rats
in the high dose and control groups.
Adrenals (s)
Epididymides (2)
Eyes with optic nerve (2)
Heart
Kidneys ( 2)
Liver (sections of two lobes)
Lungs (2)
Ovaries ( 2)
Oviducts ( 2)
Prostate
Spleen
Testes ( 2)
Uterus
Vagina

Other examinations:

No additional information available.

Statistics:

Continuous data including body weight, body weight gain, food consumption, hematology, clinical chemistry, urine analysis and absolute and relative organ weights were analyzed using analysis of variance (Snedecor and Cochran, 1967).

Reference
Snedecor, G.W. and Cochran, W.C., Statistical Methods, Iowa State University Press, Ames, Iowa, 1967, p. 215-219.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased in high dose

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Decreased in high dose

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

High dose males

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

High dose males had increased vacuolar degeneration of hepatocytes

Histopathological findings: neoplastic:

not specified

Details on results:

Observations
No mortality occurred and no treatment related effects were noted during the study. The findings that were noted such as hair loss/sore(s) around the neck/shoulders and a scaley tail were considered incidental and unrelated to dose administration.

Body Weights
Male mean body weight in the high dose group (1000 mg/kg) was statistically significantly lower than the control group throughout the treatment period (Table 1). This was considered a compound related effect. Female mean body weight was statistically significantly lower in the high dose group during the second week of study. Although not statistically significant, female mean body weight was lower than the control mean in the high dose group during the third and fourth weeks of study. This also was considered treatment related. During the second, third and fourth weeks of study, female mean body weight in the mid dose group (500 mg/kg) was slightly lower than the control mean. Based on the lower body weights observed in the high dose male and female groups, the lower body weight noted in the mid dose female group also was considered a treatment related effect.

During the third week of study, male mean body weight gain for the high dose group was lower than the control group but not statistically significant. In females, mean body weight gain in the high dose group was lower than the control group during the first week of study. During the second week of study, mean body weight gain in females was statistically significantly lower in the high dose group.

Food Consumption
Food consumption values were statistically significantly lower in males in the high dose group (1000 mg/kg) when compared with the control group during study weeks 2 and 3. During week 4 of the study, the high dose male food consumption value was lower than the control value but not statistically significant. This was considered a treatment related effect. During week -1, a statistically significant lower food consumption value was noted in the low dose group (20 mg/kg) males and was considered incidental. Although not statistically significant, the high dose female food consumption values were lower than the control group values throughout the study. These lower food consumption values correlate with the lower mean body weight and body weight gain values.

Clinical Laboratory Studies
Hematology
No compound-related effects were noted in the hematology parameters. The only statistical significance noted was a lower monocyte value in the low dose (20 mg/kg) females. This was considered a normal variation and unrelated to treatment.

Clinical Chemistry
Total protein and globulin values were statistically significantly lower in the high dose (1000 mg/kg) males when compared with the control group. The A/G ratio in males in the high dose group also was statistically significantly higher. These values may have been related to the test material, as all the high dose male rats exhibited mild vacuolar degeneration of the hepatocytes. Sodium values were statistically significantly higher in males in the low and high dose group when compared with the control group. This did not occur in a dose related fashion and the values were comparable to female values. These significant values were probably a result of the low male control value and were not considered treatment related.

Urine Analysis
No treatment-related effects were noted in any of the parameters evaluated.

Pathology
Necropsy
Various lesions were noted throughout the treated and control groups and were considered normal for rats of this strain and age. The treated males exhibited kidneys that were lighter in color than normal. This was not noted in the control groups. No effects were noted when the high dose kidneys were examined microscopically. This was not considered a compound-related effect.

Organ Weights
No compound related effects in absolute or relative organ weights were noted in any of the treated groups. A statistically significant lower absolute brain weight was noted for females in the high dose group (1000 mg/kg) when compared with the control group. This was considered an incidental finding unrelated to treatment. Brain weight relative to body weight and male absolute and relative brain weights were not statistically significant.

Microscopic Examination
Upon microscopic examination of the high dose male livers, the presence of spontaneously occurring vacuolar degeneration of the hepatocytes was more severe when compared with the control group. This finding was not observed in the female group. The difference in the intensity of this finding suggests that NIPHA may have slightly enhanced the severity of this spontaneously occurring lesion. No other compound related effects were noted when the other tissues were examined microscopically.

Dose descriptor:

NOEL

Effect level:

20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Decreased body weight observed in female rats at 500 mg/kg/day.

Dose descriptor:

NOEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Decreased body weight and food consumption observed in males at 1000 mg/kg/day. In addition, histopathologic effects were noted in the liver of male rats at 1000 mg/kg/day.

Critical effects observed:

not specified

Table 1 Summary of Body Weight Data

	Body Weights (g) at Week
Sex and Treatment Level (mg/kg)	-1	1	2	3	4
Males
Control	223 + 7	273 + 9	325 + 13	358 + 12	391 + 14
NIPHA (20 mg/kg)	223 + 4	279 + 6	334 + 5	371 + 10	404 + 9
NIPHA (500 mg/kg)	224 + 6	275 + 8	323 + 9	356 + 12	386 + 10
NIPHA (1000 mg/kg)	223 + 9	259 + 14^b	297 + 20^b	323 + 23^b	343 + 26^b
Females
Control	153 + 8	169 + 11	192 + 10	203 + 14	212 + 21
NIPHA (20 mg/kg)	153 + 10	169 + 17	192 + 19	202 + 21	214 + 24
NIPHA (500 mg/kg)	153 + 7	166 + 6	182 + 5	192 + 5	199 + 5
NIPHA (1000 mg/kg)	153 + 8	158 + 9	170 + 9^b	180 + 11	187 + 14

^a Values are group means + S.D. for 5 observations

^b Significantly different from control, p<0.05

Conclusions:

Under the conditions of this study, the no effect level of NIPHA for male rats was 500 mg/kg and for female rats, 20 mg/kg.

Executive summary:

This study was designed to evaluate the potential subchronic oral toxicity of the test article, N-isopropylhydroxylamine, NIPHA, when administered to Sprague-Dawley rats for at least 28 days. NIPHA was administered by gavage once daily to three groups of animals each composed of five male and five female rats, for at least 28 days. Dosage levels of 20, 500 and 1000 mg/kg/day were chosen for the study and were administered at a dosage volume of 10.87 ml/kg in distilled water. For comparative purposes, a concurrent vehicle control group, also composed of five male and five female rats, received distilled water on a comparable regimen at 10.87 ml/kg. The animals were observed once in the morning, one hour after dose administration and once in the afternoon for signs of overt toxicity. Detailed physical examinations, individual body weights and food consumption were recorded weekly. Clinical laboratory studies were conducted on all animals at study termination. Complete necropsy examinations were performed on all rats that were sacrificed after 28 days of dosing and organ weights obtained. Microscopic examinations were conducted on selected tissues collected for animals in the control and high dose groups.

No treatment related effects were noted in the physical observations, post-dose observations, mortality checks, clinical laboratory studies, necropsy findings or organ weights. Lower mean body weight, body weight gain and food consumption values were noted for the high dose males and females. Although not statistically significant, the mid dose females exhibited a slightly lower mean body weight when compared with the control group.

Microscopic examination of the high dose male livers revealed the presence of vacuolar degeneration of the hepatocytes. This spontaneously occurring lesion was most severe in the high dose male group when compared with the control group (1000 mg/kg). The test article may have slightly enhanced the severity of this lesion in male rats at this dose level.

Under the conditions of this study, the no effect level of NIPHA for male rats was 500 mg/kg and for female rats, 20 mg/kg.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Good

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

IPHA I-15 Hydroxylamine (IPHA) was administered by daily oral gavage to Crl: (WI) rats dosed at 20, 100 and 500 mg/kg bw/day for 90 days. Under the conditions of this study, test item related effects were observed in the high dose group; effects included death, reduced body weight gain and signs of anaemia. Spleen weight and histopathology confirmed the compensated anaemia effect in high and mid dose animals. Liver weights were increased in the high dose group of both sexes, with a similar trend in mid dose males, histopathology showed centrilobular hepatocellular vacuolation in high dose males only. Kidney weights were higher in high dose males and females, with some tubule degeneration in high dose males only. In the high dose group, there were clear signs of adverse effects. In the mid dose group the changes were minimal, but there was some evidence for a fully compensated anaemia. There were no adverse effects of treatment in the low dose group of either sex. The NOAEL (no observed adverse effect level) was considered to be 20 mg/kg bw/day.

N-isopropylhydroxylamine (NIPHA) was administered to Sprague-Dawley rats for at least 28 days. NIPHA was administered by gavage once daily to three groups of animals each composed of five male and five female rats, for at least 28 days. Dosage levels of 20, 500 and 1000 mg/kg/day were chosen for the study. No treatment related effects were noted in the physical observations, post-dose observations, mortality checks, clinical laboratory studies, necropsy findings or organ weights. Lower mean body weight, body weight gain and food consumption values were noted for the high dose males and females. Although not statistically significant, the mid dose females exhibited a slightly lower mean body weight when compared with the control group. Microscopic examination of the high dose male livers revealed the presence of vacuolar degeneration of the hepatocytes. This spontaneously occurring lesion was most severe in the high dose male group when compared with the control group (1000 mg/kg). The test article may have slightly enhanced the severity of this lesion in male rats at this dose level. Under the conditions of this study, the no effect level of NIPHA for male rats was 500 mg/kg and for female rats, 20 mg/kg.

Justification for classification or non-classification

The hemolytic anemia observed at 100 mg/kg/day does not warrant classification as STOT RE 2 as it does not meet severity criteria. At this dose-level, the hemolytic anemia is largely compensated by erythropoiesis (reticulocytes) and there are no histopathological lesions appart from (potentially hemosidrerin) pigment desposis, in the spleen only:

• Males: 11% lower RBC, 8% lower Hemoglobin, 7% lower hematocrit, 133% higher reticulocytes, 48-52% higher spleen weight, minimal to mild increased hematopoietic cells (erythtroid) in 9/10 males and minimal to mild increased numbers of pigmented (yellow) macrophages in 8/10 males.


• Females: 37% lower WBC, 26% higher reticulocytes, 10-12% higher spleen weight, minimal increased hematopoietic cells (erythtroid) in 1/10 animal and mild increased numbers of pigmented (yellow) macrophages in 2/10 rats.

RIVM’s review (Hazard classification of chemicals inducing haemolytic anaemia: an EU regulatory perpestive, 2006) gives criteria on how to apply R48/22 (now STOT RE 2) classification for hemolytic anemia. They quote:

• A hemoglobin decrease by >=20% without clear evidence for marked organ dysfunction or by >=10% with clear evidence for marked organ dysfunction. This level of effect is not met at 100 mg/kg/d (-8% hemoglobin, males only).


• Or, clear evidence for marked organ dysfunction. This notably would include, marked hemosiderosis in multiple organs. This level of effect is not met at 100 mg/kg/day (minimal to mild pigment deposit, only in spleen, no associated histopathological lesion).

The effects at 500 mg/kg/day do not need to be considered for classification since this dose-level is outside the dose-range for STOT RE 2 (10 to 100 mg/kg/d).