N-pentan-2-ylidenehydroxylamine

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

(The flow through high-concentration exposure unit was slightly below the target of 1 L/min per rat on 2 days, but still amply above the recommended minimum.This deviation was considered not to have affected the validity of the study.)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

adopted Sep 2009

Deviations:

yes

Remarks:

(The flow through high-concentration exposure unit was slightly below the target of 1 L/min per rat on 2 days, but still amply above the recommended minimum.This deviation was considered not to have affected the validity of the study.)

GLP compliance:

yes (incl. QA statement)

Remarks:

Health Care Inspectorate, Ministry of Health, Welfare and Sport, Den Haag, The Netherlands

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

484-470-6

EC Name:

-

Cas Number:

623-40-5

Molecular formula:

C5H11NO

IUPAC Name:

N-pentan-2-ylidenehydroxylamine

Test material form:

liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

For this study rats were chosen as test system, because this animal species is normally used in toxicity studies of this type and is accepted by the relevant authorities. The Sprague Dawley strain was used because it was also used in previous studies with this test material (TNO Triskelion studies 20401/13, 20451 and 20451/01)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Sprague Dawley (Crl:CD(SD)) rats were obtained from a colony maintained under specific pathogen free (SPF) conditions by Charles River Laboratories
- Age at study initiation: Approximately 8 weeks old on the day of randomization (shortly before initiation of treatment)
- Weight at study initiation: Mean body weights at the start of exposure were 322 and 204 g for male and female animals, respectively.
- Diet: Feed was provided ad libitum from the arrival of the animals until the end of the study, except during exposure and the overnight fasting period before sacrifice.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 65; Occasionally, the relative humidity briefly exceeded 65% after wet cleaning activities
- Air changes (per hr): Approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

The route of exposure to animals was inhalation. The test atmosphere in nose-only inhalation chambers, is a modification of the design of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom. The treatment groups were 0 (Vehicle control), 50, 150, 300 ppm of 2-PO for exposure period. A subset animals from control group and 300 ppm – group was exposed to ambient air during 4 weeks of recovery period. The inhalation chamber consisted of a cylindrical stainless steel column, surrounded by a transparent cylinder. The animals were secured in plastic animal holders, positioned radially through the outer cylinder around the central column. Only the nose of the rats protruded into the interior of the column. Male and female rats were placed in alternating order.

The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. To generate the test atmospheres, a liquid flow of test material, controlled by a motor driven syringe pump was allowed to evaporate in a controlled stream of humidified compressed air, by directing it through a glass evaporator which was kept at a temperature of 65.0 (±1) ˚C by circulating heated water. The resulting atmosphere was cooled by leading it through a glass coil condenser, controlled at a temperature slightly below ambient (approximately 19.5˚C) to prevent condensation of the test material in the atmosphere. All flows of air and test atmosphere were controlled and measured by mass flow controller (Bronkhorst Hi Tec, Ruurlo, The Netherlands) or mass view meter (Bronkhorst Hi Tec). Each test atmosphere was directed to the top inlet of an exposure unit, led to the noses of the animals and exhausted at the bottom of the unit. The exposure unit for the control animals was supplied with a stream of humidified compressed air only, which was measured using a mass view meter (Bronkhorst Hi Tec). The animals were placed in the exposure unit after stabilization of the test atmospheres. Test atmosphere generation and animal exposure were performed in an illuminated laboratory at room temperature. There were 10 males and 10 females per treatment groups for the exposure period. During recovery each group (control and 300 ppm-group) had 10 males and 10 females.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (Sick Maihak GMS 810 EuroFID Total Hydrocarbon Analyzer; Sick Instruments Benelux, Hedel, the Netherlands). Test atmosphere samples were taken continuously from the exposure chamber at the animals’ breathing zone and were passed to the total carbon analyzer (TCA) through a sample line. The response of the analyzers was recorded on a PC every minute using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses of the analyzers were converted to concentrations by means of calibration graphs (the formulas used to convert responses into concentrations are given below). For each exposure day, the mean concentration was calculated from the values determined every minute.

Duration of treatment / exposure:

90-day study period (65 exposure days in total)

Frequency of treatment:

6 hours/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 ppm

Remarks:

The overall average actual concentrations (± standard deviation) was 49.7 (± 0.7) ppm.

Doses / concentrations 2

Dose / conc.:

150 ppm

Remarks:

The overall average actual concentrations (± standard deviation) was 149 (± 4) ppm.

Doses / concentrations 3

Dose / conc.:

300 ppm

Remarks:

The overall average actual concentrations (± standard deviation) was 301 (± 15) ppm.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:

- Clinical signs: Animals were observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. The animals were also observed about halfway through the 6-hour exposure period, in particular to monitor any breathing abnormalities and restlessness; observation of other abnormalities was limited due to the animals’ stay in restraining tubes. All animals were thoroughly checked again in the afternoon. All abnormalities, signs of ill health, and reactions to treatment were recorded. Ophthalmoscopic observations were made prior to the start of exposure in all animals (on day -7) and towards the end of the exposure period in the animals of the control and high concentration main groups (on days 88 and 89 for females and males, respectively).

- Body weights: The body weight of each animal was recorded six (males) or seven (females) days before the start of exposure (these pre-test weights served as a basis for animal allocation), just before exposure on the first day (day 0) and twice a week thereafter (Mondays and Fridays). The animals were also weighed on the day before overnight fasting prior to necropsy, and on their scheduled sacrifice date in order to calculate the correct organ to body weight ratios.

- Clinical chemistry and haematology: Clinical chemistry was conducted at the end of the treatment period on all surviving rats of the main groups after overnight fasting, at the same time blood samples for haematology were collected. The blood was collected in heparinized plastic tubes, placed on melting ice, and plasma was prepared by centrifugation. The following measurements were made- alkaline phosphatase activity, bilirubin total, aspartate aminotransferase activity, cholesterol, alanine aminotransferase activity (ALAT) triglycerides, gamma glutamyl transferase activity (GGT), phospholipids, total protein, calcium (Ca), albumin, sodium (Na), ratio albumin to globulin, potassium (K), urea, chloride (Cl), creatinine, inorganic phosphate, fasting glucose. Since exposure-related changes were observed in animals of the main groups, investigation of clinical chemistry parameters was extended to animals of the recovery groups.

Sacrifice and pathology:

Surviving animals of the main groups were sacrificed at the end of the exposure period in such a sequence that the average time of sacrifice was approximately the same for each group. Similarly, animals of the recovery groups were sacrificed at the end of the 4-week recovery period. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes. Organs of all surviving animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Relative organ weights (g/kg body weight) were calculated from the absolute organ weight and the terminal body weight.
For histopathological examination, samples of tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of Formalin). The lungs (after weighing and processing, see above) were infused with the fixative under ca. 15 cm water pressure to ensure fixation. The carcass containing any remaining tissues was retained in formalin until completion of the histopathological examination and then discarded.

Statistics:

Appropriate statistical methods were used for analysis of data. Body weight data collected after initiation of treatment: ‘AnCova & Dunnett’s Test’ with automatic data transformation. Incidences of histopathological changes: Fisher’s exact probability test. Pre-treatment body weight, food consumption, organ weight, haematology and clinical chemistry data: ‘Generalized Anova/Ancova Test’ with automatic data transformation method. Because numerous variables were subjected to statistical analysis, the overall false positive rate (Type I errors) was greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results was based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results were significant in the light of other biological and pathological findings. Data were presented as arithmetic mean and standard deviation.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical observations revealed no treatment-related abnormalities.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male animal of the mid concentration group was humanely sacrificed on day 22 of the study, because it was suffering from a tail trauma (unrelated to the exposure to the test material).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Growth was not adversely affected by the exposure and was comparable across the groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was not adversely affected by the exposure and was comparable across the groups.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic observations revealed no treatment-related abnormalities.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Analysis of haematology parameters revealed slight changes in red blood cell and coagulation parameters of animals sacrificed at the end of the exposure period, consisting of a decreased haemoglobin concentration in males of the high concentration group (-4.4%), an increased percentage of reticulocytes (+32.5% and +28.9%) and increased numbers of thrombocytes (+18.5 and +27.9%) in males of the mid and high concentration group, and an increased prothrombin time in females of the mid and high concentration group (+5.3% and +8%). The changes were limited in magnitude, each of these changes were observed in one sex only, fully reversible within the 4-week recovery period, and not associated with any corroborative histopathological lesions. Nevertheless, they were regarded as adverse, taken into account the multiple effects in the blood and spleen related to haemolytic anaemia observed in the oral OECD 422 with 2-PO and the classification for STOT RE Cat. 2.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Investigation of clinical chemistry parameters revealed an increased plasma concentration of bilirubin in males and females of the high concentration group (+43.8% and +38.8%) sacrificed at the end of the exposure period, which was no longer observed at the end of the recovery period. Given the transient nature and the limited magnitude of this change, which was not associated with any microscopic changes, no toxicological relevance could be attached to this finding. Nevertheless, they were regarded as adverse, taken into account the multiple effects in the blood and spleen related to haemolytic anaemia observed in the oral OECD 422 with 2-PO and the classification for STOT RE Cat. 2.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant differences in absolute organ weights were observed between the groups. Organ weight data showed a statistically significant increase in the relative (to body weight) weight of the kidneys in males and females of the high concentration group, an increased relative weight of the liver in males of the mid and high concentration group, and an increase in relative spleen weight in females of the high concentration group (+13.4%) sacrificed at the end of the exposure period. The changes in relative organ weight were reversible within the 4-week recovery period and were not associated with any pathological changes.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination at scheduled termination revealed no treatment-related gross pathology.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic examination did not reveal any histopathological changes in any tissue which could be attributable to the treatment to the test material.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Effect levels

1

Key result

Dose descriptor:

NOAEC

Effect level:

149 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

organ weights and organ / body weight ratios

other: see 'Remarks'

2

Key result

Dose descriptor:

NOAEC

Remarks:

(by conversion; at 22°C and 1013 mbar)

Effect level:

615.4 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

organ weights and organ / body weight ratios

other: see 'Remarks'

Remarks on result:

other: Conversion in mg/m3 according to M. J. Derelanko, The Toxicologist's Pocket Handbook, Second Edition, 2008, Table 50 Conversion Table for Gases and Vapours

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

615.4 mg/m³ air

System:

haematopoietic

Organ:

blood

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

Exposure to the test substance resulted in slight changes in clinical pathology parameters in animals sacrificed at the end of the exposure period, (decreased haemoglobin concentration in the high-dosed males, increased percentage of reticulocytes in blood of males in the mid- and high-dose group and increased plasma bilirubin levels in males and females of the highest dose level. All changes were fully reversible after the 4-week recovery period. Investigation of other parameters did not reveal any adverse, exposure-related changes. A few statistically significant differences in coagulation parameters between animals of mid and high concentration groups versus unexposed controls (increased number of thrombocytes in males, increased prothrombin time in females) were observed. These findings were observed in one sex only (or were even reversed in the other sex), the magnitude of the changes was limited, and all changes were fully reversible within the 4-week recovery period. In the absence of any corroborative histopathological changes or significant alterations in associated parameters, a transient increase in relative weight of the kidneys (in males and females of the high concentration), liver (in males of the mid and high concentration) and spleen (in females of the high concentration group; +13.4%) were found at the end of the exposure period – but no longer at the end of the recovery period. Taken into account the multiple effects in the blood and spleen related to haemolytic anaemia observed in the oral OECD 422 with 2-PO and the classification for STOT RE Cat. 2 (Key, 2012), the described effects of this repeated dose inhalation toxicity study referring to clinical / haematological parameters and spleen weights were regarded as adverse in an overall worst-case assessment, despite the fact that they all were transient and reversible. Therefore, the medium concentration of 149 ppm (corresponding to 615.4 mg/m3) was set as No-Observed-Adverse-Effect Concentration (NOAEC) for local and systemic toxicity.