N-pentan-2-ylidenehydroxylamine

Endpoint summary

Description of key information

- LOAEL (OECD 422, oral, 28 days) = 15 mg/kg bw/day (no NOAEL could be determined)

- NOAEC (OECD 412, inhalation, 14 days) = 616.7 mg/m³

- NOAEC (OECD 413, inhalation, 90 days) = 615.4 mg/m³

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kingdom

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 71 days
- Weight at study initiation: 337 g to 394 g (males), 220 g to 271 g (females)
- Fasting period before study: none
- Housing: Animals were housed inside a barriered rodent facility. The gridded cages used during pairing were suspended over trays covered with absorbent paper which was changed daily. For cages with solid floors, wood based material was used as bedding and was sterilised by autoclaving and changed at least twice each week. Cages, cage-trays, food hoppers and water bottles were changed at appropriate intervals. The cages were distributed on the racking to equalise, as far as possible, environmental influences amongst the groups.
- Diet: ad libitum (SDS VRF1 Certifie Diet) except overnight before routine blood sampling. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: ad libitum from the public supply via polycarbonate or polypropylene bottles fitted with sipper tubes.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23
- Humidity (%): 40 to 70
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: To: 21 March 2012 to 23 May 2012

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance, MPKO, was prepared for administration as a series of graded concentrations in the vehicle, by dilution of individual weighings of the test substance. Small amounts of vehicle were added to the test substance and mixed until a solution was formed. This was made up to the required volume with vehicle and then magnetically stirred until homogenous. The test substance was used as supplied. All formulations were prepared weekly and stored refrigerated before use.


VEHICLE
- Concentration in vehicle: 7.5, 25, 75 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before treatment commenced, the suitability of the proposed mixing procedure was determined and specimen formulations were analysed to assess the homogeneity and stability of the test material in the liquid matrix. Specimen formulations (typically 400 mL) were prepared at concentrations of 1 mg/mL and 100 mg/mL and equally split between four amber screw-capped bottles. Prior to initial sampling on each day, the formulation was mixed by 20-fold inversion. A control vehicle sample was stored with each batch of stability samples. The stability was confirmed for at least 24 hours at ambient temperature and for up to 15 days when refrigerated (2-8°C).
Samples of each formulation prepared for administration on the first and last occasion of treatment were analysed for achieved concentration of the test substance. Four samples were taken (nominally 1 mL accurately weighed) from all groups. Two of the samples from each group were analysed. The remainder were retained as contingency for analysis if any result required confirmation.

Duration of treatment / exposure:

The test substance, MPKO, was administered for two weeks before pairing up to necropsy (at least five weeks) for males and two weeks before pairing then throughout pairing and gestation until Day 6 of lactation for females. Recovery animals were treated for approximately six weeks and completed a further 14 days without treatment.

Animals of the F1 generation were not dosed.

Frequency of treatment:

All animals were dosed once each day at approximately the same time each day, seven days per week.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 males per group (Groups 1 to 4)
15 females (Groups 1 and 4), of these 5 females per group were not mated and were used for the recovery group
10 females (Groups 2 and 3)

Control animals:

yes, concurrent vehicle

Details on study design:

Ten males and 10 females per group were treated for two weeks at dose levels of 15, 50 or 150 mg/kg/day before pairing. Treatment continued to a total of at least 5 weeks. A control group of 10 male and 10 female rats received the vehicle, corn oil, at the same volume-dose throughout the same period. Males were killed after at least 5 weeks of treatment and females were killed on Day 7 of lactation.
Recovery, over 14 days without treatment, was assessed in five of the control and five of the high dose males and in an extra five unmated females in the same groups which were treated for 6 weeks before start of recovery.
The F1 generation received no direct administration of the test substance; any exposure was in utero or via the milk.
During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, gestation length and parturition observations, haematology, blood chemistry, organ weight, macropathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily during the first week of treatment and weekly thereafter for all animals and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation for mated females only

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before treatment commenced and during each week of treatment and recovery periods for each adult and on Days 0, 6, 13 and 20 after mating and Days 1 and 6 of lactation for Main study females only.

BODY WEIGHT: Yes
- Time schedule for examinations: On the day that treatment commenced (Week 0), weekly thereafter for the treatment and recovery periods and before necropsy. The weight of each Main study female was also recorded on Days 0, 6, 13 and 20 after mating and on Days 1, 4 and 7 of lactation.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded on a weekly basis. The males had no food consumption performed in Week 3 due to pairing. The Main study females were recorded on a weekly basis until they were paired for mating. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.
For each Main study female, the weight of food supplied, that remaining and an estimate of any spilled was also recorded for the periods Days 0-5, 6-12 and 13-19 after mating and Days 1-3 and 4-6 of lactation. From these records the mean daily consumption (g/rat/day) was calculated for each animal.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 2 and Recovery Week 2
- Anaesthetic used for blood collection: Yes, isofluorane
- Animals fasted: Yes
- How many animals: 5/sex
- Parameters checked included thoses listed in the OECD guidance.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 2 and Recovery Week 2
- Animals fasted: Yes
- How many animals: 5/sex
- Parameters checked included those listed int eh OECD guidance.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 5 (males), Days 4-6 of lactation (females)
- Dose groups that were examined: Groups 1, 2, 3, and 4
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

All statistical analyses were carried out separately for males and females. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment with MPKO at 50 and 150 mg/kg bw/day was associated with major adverse effects upon the red blood cells. Many of the affected paramters showed complete recovery after the 14-day recovery period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Several parameters at 50 and 150 mg/kg/day were affected in males and/or females during Week 2 of the treatment period. The affected parameters were similar to control after the 14-day recovery period.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Increased spleen weights in males and females at >= 50 mg/kg bw/day and increased heart weight (slight) in females at 150 mg/kg bw/day. After the 14-day recovery period, splpeen weights were still slightly higer in the 150 mg/kg bw/day males and females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged (>=50 mg/kg bw/day) and dark (>=15 mg/kg bw/day) colored spleens in males and females. Dark colored kidneys in females at >=50 mg/kg bw/day. The dark colored spleens were also observed at 150 mg/kg bw/day in recovery animals.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Spleen: Both sexes - haemosiderosis >=15 mg/kg bw/day, congestion >=50 mg/kg bw/day, extramedullary haematopoiesis 150 mg/kg bw/day. Liver: Both sexes - extramedullary haematopoiesis at 150 mg/kg bw/day. Full recovery of extramedullary haematopoiesis.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths and no post dosing signs.

BODY WEIGHT AND WEIGHT GAIN
There were no adverse bodyweight effects in males or females before or after mating, during lactation and during recovery.

FOOD CONSUMPTION
Food consumption was not affected by treatment with MPKO.

HAEMATOLOGY
Treatment with MPKO at 50 and 150 mg/kg bw/day was associated with major adverse effects upon the red blood cells and the following changes were attributed to treatment. Males and females receiving 50 and 150 mg/kg bw/day showed low haematocrit (-12 and -17% (m); -9.6% and -11% (f)) and haemoglobin levels (-15.2% and -22% (m); -12% and -16.7% (f)), low red blood cell counts (-15.5% and -31.5% (m);-12.1% and -29.2% (f)), high reticulocytes (+184% and +383% (m); +159% and +346% (f)) and low mean cell haemoglobin concentrations (-3.6% and -6.4% (m); -2.7% and -6.3% (f)) compared to control, and for males only at those dose levels high mean cell volumes (+4.5% and +21%). A dose relationship was also apparent. The high mean cell volume in females was restricted to those receiving 150 mg/kg/day(+26.5%). High mean cell haemoglobin levels (+13.2% (m); +18.1% (f)) and high platelet levels (+40.8% (m) and +23.3% (f)) in animals receiving 150 mg/kg bw/day were also evident.

After the 14-day off dose period many of the parameters noted to be different to control during treatment had shown complete recovery. Those changes which had not completely resolved included high mean cell haemoglobin level (+11.2%), low mean cell haemoglobin concentration (-2.8%) and high mean cell volumes (+13.5%) for males previously receiving 150 mg/kg bw/day, low red blood cell counts (-7.8%), high mean cell haemoglobin level (+12.2%) and high mean cell volume (+13.4%) for females previously receiving the same dose.

CLINICAL CHEMISTRY
High bilirubin concentrations were recorded for males and females at 50 and 150 mg/kg bw/day during the week 2 of the treatment period. There was a suggestion of an increase in potassium and phosphorus in males receiving 150 mg/kg bw/day. Total protein was low for males attaining statistical significance for those receiving 50 and 150 mg/kg/day and albumin for males at 150 mg/kg bw/day was also low. Females receiving 150 mg/kg bw/day had a high Albumin/Globulin ratio.

The affected parameters were similar to control following two weeks of recovery.

NEUROBEHAVIOUR
Sensory reactivity findings and grip strength values for males and females were unaffected by treatment with MPKO.
There was no effect of MPKO on motor activity scores.

PRE-COITAL INTERVAL
All animals mated within the first four days following pairing

MATING PERFORMANCE AND FERTILITY
Percentage mating, conception rate and fertility index scores for all groups were 100%.

GESTATION LENGTH, PARTURITION, AND GESTATION INDEX
Gestation length was within the expected range for this strain for of rat at these laboratories. Two females, one each receiving 50 and 150 mg/kg bw/day, found to have implantation scars at necropsy on Day 25 of gestation but were not observed to give birth either due to total litter resorption or cannibalising pups born overnight prior to the first check of the day: a relationship to treatment is not inferred.

ORGAN WEIGHTS
Adjusted mean spleen weight was higher than control in males (+59.9% and +218%) and females (+25.4% and +147.2%) receiving 50 or 150 mg/kg bw/day with a dose response evident. In females receiving 150 mg/kg bw/day there was a slight increase in adjusted mean heart weight.

After 14 days recovery, the spleen weights were still slightly higher than Control values for males and females which had received 150 mg/kg bw/day; although no statistical significance was attained for females, and the values were lower than the main study animals. The heart weights of females receiving 150 mg/kg bw/day were similar to control values. Kidney weights were slightly higher than Control after the recovery period in males that had received 150 mg/kg bw/day this was not seen in the main phase animals.

GROSS PATHOLOGY
Enlarged spleen was seen in all males and females treated with 150 mg/kg bw/day and in three out of 10 males treated with 50 mg/kg bw/day. Dark colouration of the spleen was also seen in all males and females treated with 150 mg/kg bw/day, in nine out of 10 males and females treated with 50 mg/kg bw/day and in one out of 10 males treated with 15 mg/kg bw/day.

Dark colouration of the kidneys (left and right) was seen in nine females treated with 150 mg/kg bw/day and in three females treated with 50 mg/kg bw/day.

After the recovery period, dark colouration of the spleen was seen in three males and two females treated with 150 mg/kg bw/day

HISTOPATHOLOGY: NON-NEOPLASTIC
Spleen: Haemosiderosis was observed in males and females treated at 15, 50 and 150 mg/kg bw/day. Congestion was also seen in males and females treated at 50 and 150 mg/kg bw/day. An increase in the incidence of extramedullary haemopoiesis was also observed in males and females treated at 150 mg/kg bw/day. These changes revealed dose-relationship.
Liver: Extramedullary haemopoiesis was observed in males and females treated at 50 and 150 mg/kg bw/day.

After Recovery Period:
Spleen: An increase in the severity of haemosiderosis was observed in males and females previously treated at 150 mg/kg bw/day. A decrease in severity of congestion was also observed in males previously treated at 150 mg/kg bw/day.

Key result

Dose descriptor:

LOAEL

Remarks:

(toxicity to F0 generation)

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Remarks:

(developmental/reproductive effects)

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: There were no effects on any of the reproductive parameters or developmental endpoints evaluated.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

haematopoietic

Organ:

blood

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Due to the multiple effects in the blood and spleen related to haemolytic anaemia 2-PO is classified for STOT RE Cat 2.

Executive summary:

The effects observed in this study included changes in haematological parameters and organ weights starting at a dose level of 50 mg/kg bw/day and above and in microscopic tissue appearance (haemosiderosis) at dose levels of 15 mg/kg bw/day and above. After two weeks of dose complete recovery was seen in many clinical pathology parameters and recovery was in progress but not complete in males for high mean cell haemoglobin level, low mean cell haemoglobin concentration and high mean cell volume and in females for low red blood cell counts, high mean cell haemoglobin level and high mean cell volume, organ weights, macroscopic and microscopic appearance. There were no adverse effects of treatment on the reproductive/developmental screening parameters assessed at least 150 mg/kg bw/day.

The Low Observed Adverse Effect Level (LOAEL) for 2-PO (MPKO) is considered to be 15 mg/kg bw/day for general systemic toxicity (the lowest dose tested), taken into account that haemosiderosis, which is regarded as an adverse effect related to haemolytic anaemia, was already observed in the lowest dose group of 15 mg/kg bw/day. At the two higher dose groups multiple effects related to haemolytic anaemia developed, showing the dose-dependent progress of effects related to haemolytic anaemia. The NOAEL for reproductive and developmental screening parameters is considered to be 150 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

15 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

System:

haematopoietic

Organ:

blood

spleen

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

The temperature and relative humidity in the exposure unit or animal room occasionally deviated from target limits. These deviations were 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

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: colony maintained under specific pathogen-free (SPF) conditions at Charles River (Sulzfeld, Germany)
- Age at study initiation: 8 weeks
- Weight at study initiation: mean body weights: 328 g for male and 205 g for female animals
- Housing: under conventional conditions separated by sex, in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment
- Diet (e.g. ad libitum): cereal-based rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3)
ad libitum from the arrival of the animals until the end of the study, except during inhalation exposure and during the fasting period prior to the collection of blood for clinical pathology
- Water (e.g. ad libitum): domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed.
ad libitum from the arrival of the animals until the end of the study, except during inhalation exposure and during the fasting period prior to the collection of blood for clinical pathology
- Acclimation period: 7 days

IN-LIFE DATES: From: 2014-01-22--24 To: 2014-02-05--07 depending on the groups.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): Lighting was artificial (fluorescent tubes) with a sequence of 12 hours light and 12 hours dark.

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

EXPOSURE APPARATUS:
nose-only inhalation chambers (each group in a separate chamber; chamber types; groups 2-4: a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom; group 1: chamber manufactured by P. Groenendijk Kunststoffen B.V., the Netherlands; see Figure 1). The inhalation chamber consisted of a cylindrical column of aluminum (groups 2-4) or polypropylene (group 1), surrounded by a transparent cylinder.
VOLUME COLUMN: 39 (group 1) or 37 litres (groups 2-4)
DETAILS COLUMN: top assembly with the entrance of the unit, one mixing chamber, a rodent tube section, and at the bottom the base assembly with the exhaust port.
METHOD OF HOLDING ANIMALS IN TEST CHAMBER:
The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column.
TOTAL AIRFLOW THROUGH UNIT:
at least 1 litre/min per animal

GENERATION TEST ATMOSPHERE:
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 (WPI Type SP220i, World Precision Instruments, Sarasota FL, USA), was allowed to evaporate in a mass flow controlled stream of humidified air, by directing it through a glass evaporator at 65.0 ̊C. The resulting atmosphere was cooled by leading it through a coil condenser which was controlled at 19 ̊C. The vapour was transported in a stream of humidified compressed air, the flow of which was controlled by means of a mass flow controller (Bronkhorst, Hi Tec, Ruurlo, The Netherlands).
The test atmospheres for the mid concentration and high concentration groups were generated separately (i.e. by using a separate syringe pump, evaporator and condensor for these concentrations). The test atmosphere for the low concentration group was obtained by diluting the high-concentration test atmosphere. For this purpose, a mass flow controlled stream of the high-concentration atmosphere was supplemented with a mass flow controlled stream of humidified compressed air via an eductor (Fox Eductor from Fox Valve Development Corp., Dover, NJ, USA). 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 measured stream of humidified compressed air only.
The animals were placed in the exposure unit after stabilization of the test atmosphere.

TEMPERATURE, HUMUDITY, PRESSURE IN AIR CHAMBER: The chamber airflow of the test atmospheres was recorded about hourly by means of the settings of the flow controllers. The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). The concentrations of oxygen (oxygen analyser type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70, Vaisala, Helsinki, Finland) in the test atmosphere were measured during exposure on the first exposure day.

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. The response of the analyser was recorded on a PC every minute using a CAN transmitter. The responses of the analysers were converted to concentrations by means of calibration graphs. For each exposure day, the mean concentration was calculated from the values determined every minute. Representative test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were passed to the total carbon analyser (TCA) through a sample line.

Duration of treatment / exposure:

10 days

Frequency of treatment:

6 hours/day - 5days/week

Dose / conc.:

52.9 ppm (analytical)

Remarks:

+/- 2.8 ppm

Dose / conc.:

149.3 ppm (analytical)

Remarks:

+/- 1.9 ppm

Dose / conc.:

289.9 ppm (analytical)

Remarks:

+/- 3.1 ppm

No. of animals per sex per dose:

5

Control animals:

yes

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT:
- Time schedule for examinations: before start of exposure and then twice weekly

FOOD CONSUMPTION:
- Food consumption was measured over two 7-day periods (males) or a 7-day period followed by a 6-day period (females), starting on day 0.
The results were expressed in g per animal per day.

BLOOD SAMPLE COLLECTION
To enable possible future determination of the concentration of test material in blood, two blood samples were collected from male animals of the control group and the high-concentration group. Heparin was used as anticoagulant. The samples were stored frozen (-70 ̊C).

HAEMATOLOGY:
-Time: at the end of the treatment period (one or two days before scheduled sacrifice)
- Animals: on all animals
-Fasting: overnight fasting (water was freely available).
- Anaesthesia: CO2/O2 or pentobarbital anaesthesia
- Anticoagulant: EDTA
-Parameters checked: red blood cells (RBC), haemoglobin (Hb), packed cell volume (PCV) reticulocytes, total white blood cells (WBC) differential white blood cells1 prothrombin time (PT) thrombocytes

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: collected from fasted animals in the same way and at the same time as the samples for haematology
- Anticoagulant: heparin
- Parameters checked: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), bilirubin (total), total protein, albumin, cholesterol (total), phospholipids, triglycerides, creatinine, urea, inorganic phosphate (PO4), calcium (Ca), ratio albumin to globulin (calculated), glucose, chloride (Cl), potassium (K), sodium (Na)

ORGAN WEIGHTS
- At scheduled necropsy of the animals, as soon as possible after dissection
- Preservation: 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4%)
- Organs: adrenals, brain, heart, kidneys, liver, lung with trachea and larynx, ovaries, spleen, testes, thymus, thyroid, uterus

Sacrifice and pathology:

SACRIFICE
- Animals were sacrificed on day 14, after overnight fasting, by exsanguination from the abdominal aorta. The animals were anaesthetized by intraperitoneal injection of sodium pentobarbital.

HISTOPATHOLOGICAL EXAMINATION:
- embedded in paraffin wax
- sectioned at 5 μm
- stained with haematoxylin and eosin
- animals: low- and mid- concentration groups were not processed, except for the nose which was decalcified and embedded in paraffin concurrently with the nose of the control animals and the high concentration group
- Histopathological examination: light microscopy on all tissues and organs listed above. In addition, all gross lesions were examined in the low- and mid-concentration groups.

Statistics:

Tests were performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.
Statistical significance was determined with An(c)ova, Kruskal-Wallis and Dunnett's test.
Details regarding the statistical analysis of the results is presented in the section 'any other information on materials and methods'.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

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

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

CLINICAL OBSERVATION AND MORTALITY
All animals survived until scheduled sacrifice. No abnormalities were observed in animals exposed to the test material or to clean air. No abnormalities were seen at the group-wise observations made about halfway each 6-hour exposure period.

BODY WEIGHT
All animals gained comparable body weight during the 14-day exposure period. Mean body weights of animals exposed to the test material were similar to the control (clean air) body weights throughout the study.

FOOD CONSUMPTION
Food consumption was not affected by the exposure to the test material.

HAEMATOLOGY
Red blood cell and coagulation values and total and differential white blood cell values showed no treatment-related changes. A few statistically significant differences between animals exposed to the test material and the clean air controls occurred but these findings were not ascribed to treatment, namely:
- Higher mean corpuscular haemoglobin (MCH) in females of the low concentration group. There was no concentration-related response. Moreover, the measured red blood cell parameters (red blood cell count, haemoglobin or packed cell volume) showed no significant changes.
Therefore, the difference in the calculated parameter MCH was considered to be a chance finding.
- Higher number of thrombocytes in males of the mid-concentration group. This difference was not ascribed to treatment because there was no concentration- related response.
- Higher percentage of lymphocytes and lower percentage and absolute number of neutrophils in females of the low- and high-concentration groups. In the absence of a concentration-related response these differences were considered to be chance findings.

CLINICAL CHEMISTRY
Clinical chemistry results showed no treatment-related changes. The few statistical significances observed were considered to be unrelated to treatment because the data showed no concentration-related response (males: higher albumin/globulin ratio in the low- and high-concentration groups, higher calcium in the mid- concentration group; females: lower ASAT in all exposed groups, lower albumin/globulin ratio in the low- and mid-concentration groups).

ORGAN WEIGHTS
The organ weight results showed the following statistically significant differences between animals exposed to the test material and clean air controls:
- Higher spleen weight (absolute and relative to body weight) in males of the mid-and high-concentration groups compared to control group animals (absolute: +13.2% and +17.2%; relative: +17.2% and 17.2%).
- Higher weight of the ovaries (absolute and relative to body weight) in females of the mid-concentration group compared to control group animals. This finding was not ascribed to treatment because there was no concentration-related response.

PATHOLOGY
-Macroscopic examination: There were no macroscopic findings attributable to the exposure to the test material. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally.
-Microscopic examination: Microscopic examination did not reveal treatment-related histopathological changes. The few histopathological changes observed in the high-concentration group were considered unremarkable because they represented background findings and occurred in only one or two animals or at about the same incidence in the high-concentration group and the control group.

Dose descriptor:

NOAEC

Effect level:

149.3 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEC

Effect level:

616.7 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

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

Critical effects observed:

yes

Lowest effective dose / conc.:

616.7 mg/m³ air

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Monitoring of exposure conditions:

1. ACTUAL CONCENTRATION:

The overall mean actual concentrations (+/- standard deviation) of the test material in the test atmospheres as measured by total carbon analysis were 52.9 (+/- 2.8), 149.3 (+/- 1.9) and 298.9 (+/- 3.1) ppm for the low-, mid- and high-concentration. These were close to the target concentrations (50, 150 and 300 ppm).

2. TIME TO ATTAIN CHAMBER EQUILIBRATION:

The time to reach 95% of the steady state concentration (T95) was calculated to be about 6 minutes (based on a chamber volume of 37 L and airflow of about 20 L/min).

3. NOMINAL CONCENTRATION AND GENERATION EFFICIENCY:

The mean nominal concentrations (+/- standard deviation) were 52.6 (+/- 2.06), 163.5 (+/- 3.35) and 309.6 (+/- 4.01) ppm for the low-, mid- and high-concentration, indicating generation efficiencies of 100.8%, 91.4% and 96.6%, respectively.

4. AIRFLOW, TEMPERATURE AND RELATIVE HUMIDITY:

The overall mean (± standard deviation) chamber airflows were 20.2 (± 0.19), 23.2 (± 0.27), 20.2 (± 0.18) and 23.5 (± 0.24) L/min for the control, low, mid and high exposure groups respectively. The air temperature in the exposure chambers during exposure was generally within the target range of 20–24°C, minor excursions outside the lower end occurred for the control, low and mid exposure groups. The overall mean temperature was 21.4, 21.6, 21.7 and 21.9°C for chambers of control, low, mid and high exposure groups. The relative humidity during exposure was generally within the target range of 30- 70%, minor excursions outside the lower end occurred for groups control, mid and high. The overall mean relative humidity was 39.3, 40.2, 36.1 and 34.9% in chambers of control, low, mid and high exposure groups. The oxygen concentration, measured on the first exposure day, was 20.2% for exposure chamber control, 20.3% for chamber low, 20.5% for chamber mid and 20.6% for chamber high. The carbon dioxide concentration in the test atmospheres, measured on the first exposure day, was 0.491 vol% for exposure chamber control, 0.418 vol% for chamber low, 0.236 vol% for chamber mid and 0.199 vol% for chamber high

Conclusions:

Under the conditions of this 14-day study exposure to 2-PO by inhalation at concentrations up to 298.9 ppm (actual concentration; 6 hours/day, 5 days/week) was tolerated without obvious signs of toxicity. The only finding were higher spleen weights (absolute and relative to body weight) in males of the mid-and high-concentration groups compared to control group animals (absolute: +13.2% and +17.2%; relative: +17.2% and 17.2%). This increase in absolute and relative spleen weights of male animals, starting at the mid dose group, 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. Therefore, 149.3 ppm (corresponding to 616.7 mg/m3), was determined to be the No-Observed-Adverse-Effect Concentration (NOAEC) for local and systemic toxicity.

Reference 2

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.)

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

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

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

Dose / conc.:

50 ppm

Remarks:

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

Dose / conc.:

150 ppm

Remarks:

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

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

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.

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

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'

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

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

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

615.4 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises two adequate and reliable key studies, and are thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

System:

haematopoietic

Organ:

blood

spleen

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

The temperature and relative humidity in the exposure unit or animal room occasionally deviated from target limits. These deviations were 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

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: colony maintained under specific pathogen-free (SPF) conditions at Charles River (Sulzfeld, Germany)
- Age at study initiation: 8 weeks
- Weight at study initiation: mean body weights: 328 g for male and 205 g for female animals
- Housing: under conventional conditions separated by sex, in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment
- Diet (e.g. ad libitum): cereal-based rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3)
ad libitum from the arrival of the animals until the end of the study, except during inhalation exposure and during the fasting period prior to the collection of blood for clinical pathology
- Water (e.g. ad libitum): domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed.
ad libitum from the arrival of the animals until the end of the study, except during inhalation exposure and during the fasting period prior to the collection of blood for clinical pathology
- Acclimation period: 7 days

IN-LIFE DATES: From: 2014-01-22--24 To: 2014-02-05--07 depending on the groups.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45 - 65%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): Lighting was artificial (fluorescent tubes) with a sequence of 12 hours light and 12 hours dark.

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

EXPOSURE APPARATUS:
nose-only inhalation chambers (each group in a separate chamber; chamber types; groups 2-4: a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom; group 1: chamber manufactured by P. Groenendijk Kunststoffen B.V., the Netherlands; see Figure 1). The inhalation chamber consisted of a cylindrical column of aluminum (groups 2-4) or polypropylene (group 1), surrounded by a transparent cylinder.
VOLUME COLUMN: 39 (group 1) or 37 litres (groups 2-4)
DETAILS COLUMN: top assembly with the entrance of the unit, one mixing chamber, a rodent tube section, and at the bottom the base assembly with the exhaust port.
METHOD OF HOLDING ANIMALS IN TEST CHAMBER:
The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column.
TOTAL AIRFLOW THROUGH UNIT:
at least 1 litre/min per animal

GENERATION TEST ATMOSPHERE:
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 (WPI Type SP220i, World Precision Instruments, Sarasota FL, USA), was allowed to evaporate in a mass flow controlled stream of humidified air, by directing it through a glass evaporator at 65.0 ̊C. The resulting atmosphere was cooled by leading it through a coil condenser which was controlled at 19 ̊C. The vapour was transported in a stream of humidified compressed air, the flow of which was controlled by means of a mass flow controller (Bronkhorst, Hi Tec, Ruurlo, The Netherlands).
The test atmospheres for the mid concentration and high concentration groups were generated separately (i.e. by using a separate syringe pump, evaporator and condensor for these concentrations). The test atmosphere for the low concentration group was obtained by diluting the high-concentration test atmosphere. For this purpose, a mass flow controlled stream of the high-concentration atmosphere was supplemented with a mass flow controlled stream of humidified compressed air via an eductor (Fox Eductor from Fox Valve Development Corp., Dover, NJ, USA). 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 measured stream of humidified compressed air only.
The animals were placed in the exposure unit after stabilization of the test atmosphere.

TEMPERATURE, HUMUDITY, PRESSURE IN AIR CHAMBER: The chamber airflow of the test atmospheres was recorded about hourly by means of the settings of the flow controllers. The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). The concentrations of oxygen (oxygen analyser type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70, Vaisala, Helsinki, Finland) in the test atmosphere were measured during exposure on the first exposure day.

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. The response of the analyser was recorded on a PC every minute using a CAN transmitter. The responses of the analysers were converted to concentrations by means of calibration graphs. For each exposure day, the mean concentration was calculated from the values determined every minute. Representative test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were passed to the total carbon analyser (TCA) through a sample line.

Duration of treatment / exposure:

10 days

Frequency of treatment:

6 hours/day - 5days/week

Dose / conc.:

52.9 ppm (analytical)

Remarks:

+/- 2.8 ppm

Dose / conc.:

149.3 ppm (analytical)

Remarks:

+/- 1.9 ppm

Dose / conc.:

289.9 ppm (analytical)

Remarks:

+/- 3.1 ppm

No. of animals per sex per dose:

5

Control animals:

yes

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: daily

BODY WEIGHT:
- Time schedule for examinations: before start of exposure and then twice weekly

FOOD CONSUMPTION:
- Food consumption was measured over two 7-day periods (males) or a 7-day period followed by a 6-day period (females), starting on day 0.
The results were expressed in g per animal per day.

BLOOD SAMPLE COLLECTION
To enable possible future determination of the concentration of test material in blood, two blood samples were collected from male animals of the control group and the high-concentration group. Heparin was used as anticoagulant. The samples were stored frozen (-70 ̊C).

HAEMATOLOGY:
-Time: at the end of the treatment period (one or two days before scheduled sacrifice)
- Animals: on all animals
-Fasting: overnight fasting (water was freely available).
- Anaesthesia: CO2/O2 or pentobarbital anaesthesia
- Anticoagulant: EDTA
-Parameters checked: red blood cells (RBC), haemoglobin (Hb), packed cell volume (PCV) reticulocytes, total white blood cells (WBC) differential white blood cells1 prothrombin time (PT) thrombocytes

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: collected from fasted animals in the same way and at the same time as the samples for haematology
- Anticoagulant: heparin
- Parameters checked: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), bilirubin (total), total protein, albumin, cholesterol (total), phospholipids, triglycerides, creatinine, urea, inorganic phosphate (PO4), calcium (Ca), ratio albumin to globulin (calculated), glucose, chloride (Cl), potassium (K), sodium (Na)

ORGAN WEIGHTS
- At scheduled necropsy of the animals, as soon as possible after dissection
- Preservation: 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4%)
- Organs: adrenals, brain, heart, kidneys, liver, lung with trachea and larynx, ovaries, spleen, testes, thymus, thyroid, uterus

Sacrifice and pathology:

SACRIFICE
- Animals were sacrificed on day 14, after overnight fasting, by exsanguination from the abdominal aorta. The animals were anaesthetized by intraperitoneal injection of sodium pentobarbital.

HISTOPATHOLOGICAL EXAMINATION:
- embedded in paraffin wax
- sectioned at 5 μm
- stained with haematoxylin and eosin
- animals: low- and mid- concentration groups were not processed, except for the nose which was decalcified and embedded in paraffin concurrently with the nose of the control animals and the high concentration group
- Histopathological examination: light microscopy on all tissues and organs listed above. In addition, all gross lesions were examined in the low- and mid-concentration groups.

Statistics:

Tests were performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.
Statistical significance was determined with An(c)ova, Kruskal-Wallis and Dunnett's test.
Details regarding the statistical analysis of the results is presented in the section 'any other information on materials and methods'.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

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

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Details on results:

CLINICAL OBSERVATION AND MORTALITY
All animals survived until scheduled sacrifice. No abnormalities were observed in animals exposed to the test material or to clean air. No abnormalities were seen at the group-wise observations made about halfway each 6-hour exposure period.

BODY WEIGHT
All animals gained comparable body weight during the 14-day exposure period. Mean body weights of animals exposed to the test material were similar to the control (clean air) body weights throughout the study.

FOOD CONSUMPTION
Food consumption was not affected by the exposure to the test material.

HAEMATOLOGY
Red blood cell and coagulation values and total and differential white blood cell values showed no treatment-related changes. A few statistically significant differences between animals exposed to the test material and the clean air controls occurred but these findings were not ascribed to treatment, namely:
- Higher mean corpuscular haemoglobin (MCH) in females of the low concentration group. There was no concentration-related response. Moreover, the measured red blood cell parameters (red blood cell count, haemoglobin or packed cell volume) showed no significant changes.
Therefore, the difference in the calculated parameter MCH was considered to be a chance finding.
- Higher number of thrombocytes in males of the mid-concentration group. This difference was not ascribed to treatment because there was no concentration- related response.
- Higher percentage of lymphocytes and lower percentage and absolute number of neutrophils in females of the low- and high-concentration groups. In the absence of a concentration-related response these differences were considered to be chance findings.

CLINICAL CHEMISTRY
Clinical chemistry results showed no treatment-related changes. The few statistical significances observed were considered to be unrelated to treatment because the data showed no concentration-related response (males: higher albumin/globulin ratio in the low- and high-concentration groups, higher calcium in the mid- concentration group; females: lower ASAT in all exposed groups, lower albumin/globulin ratio in the low- and mid-concentration groups).

ORGAN WEIGHTS
The organ weight results showed the following statistically significant differences between animals exposed to the test material and clean air controls:
- Higher spleen weight (absolute and relative to body weight) in males of the mid-and high-concentration groups compared to control group animals (absolute: +13.2% and +17.2%; relative: +17.2% and 17.2%).
- Higher weight of the ovaries (absolute and relative to body weight) in females of the mid-concentration group compared to control group animals. This finding was not ascribed to treatment because there was no concentration-related response.

PATHOLOGY
-Macroscopic examination: There were no macroscopic findings attributable to the exposure to the test material. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally.
-Microscopic examination: Microscopic examination did not reveal treatment-related histopathological changes. The few histopathological changes observed in the high-concentration group were considered unremarkable because they represented background findings and occurred in only one or two animals or at about the same incidence in the high-concentration group and the control group.

Dose descriptor:

NOAEC

Effect level:

149.3 ppm (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Dose descriptor:

NOAEC

Effect level:

616.7 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

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

Critical effects observed:

yes

Lowest effective dose / conc.:

616.7 mg/m³ air

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Monitoring of exposure conditions:

1. ACTUAL CONCENTRATION:

The overall mean actual concentrations (+/- standard deviation) of the test material in the test atmospheres as measured by total carbon analysis were 52.9 (+/- 2.8), 149.3 (+/- 1.9) and 298.9 (+/- 3.1) ppm for the low-, mid- and high-concentration. These were close to the target concentrations (50, 150 and 300 ppm).

2. TIME TO ATTAIN CHAMBER EQUILIBRATION:

The time to reach 95% of the steady state concentration (T95) was calculated to be about 6 minutes (based on a chamber volume of 37 L and airflow of about 20 L/min).

3. NOMINAL CONCENTRATION AND GENERATION EFFICIENCY:

The mean nominal concentrations (+/- standard deviation) were 52.6 (+/- 2.06), 163.5 (+/- 3.35) and 309.6 (+/- 4.01) ppm for the low-, mid- and high-concentration, indicating generation efficiencies of 100.8%, 91.4% and 96.6%, respectively.

4. AIRFLOW, TEMPERATURE AND RELATIVE HUMIDITY:

The overall mean (± standard deviation) chamber airflows were 20.2 (± 0.19), 23.2 (± 0.27), 20.2 (± 0.18) and 23.5 (± 0.24) L/min for the control, low, mid and high exposure groups respectively. The air temperature in the exposure chambers during exposure was generally within the target range of 20–24°C, minor excursions outside the lower end occurred for the control, low and mid exposure groups. The overall mean temperature was 21.4, 21.6, 21.7 and 21.9°C for chambers of control, low, mid and high exposure groups. The relative humidity during exposure was generally within the target range of 30- 70%, minor excursions outside the lower end occurred for groups control, mid and high. The overall mean relative humidity was 39.3, 40.2, 36.1 and 34.9% in chambers of control, low, mid and high exposure groups. The oxygen concentration, measured on the first exposure day, was 20.2% for exposure chamber control, 20.3% for chamber low, 20.5% for chamber mid and 20.6% for chamber high. The carbon dioxide concentration in the test atmospheres, measured on the first exposure day, was 0.491 vol% for exposure chamber control, 0.418 vol% for chamber low, 0.236 vol% for chamber mid and 0.199 vol% for chamber high

Conclusions:

Under the conditions of this 14-day study exposure to 2-PO by inhalation at concentrations up to 298.9 ppm (actual concentration; 6 hours/day, 5 days/week) was tolerated without obvious signs of toxicity. The only finding were higher spleen weights (absolute and relative to body weight) in males of the mid-and high-concentration groups compared to control group animals (absolute: +13.2% and +17.2%; relative: +17.2% and 17.2%). This increase in absolute and relative spleen weights of male animals, starting at the mid dose group, 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. Therefore, 149.3 ppm (corresponding to 616.7 mg/m3), was determined to be the No-Observed-Adverse-Effect Concentration (NOAEC) for local and systemic toxicity.

Reference 2

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.)

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

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

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

Dose / conc.:

50 ppm

Remarks:

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

Dose / conc.:

150 ppm

Remarks:

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

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

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.

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

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'

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

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 243.1 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises two adequate and reliable key studies, and are thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

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

There are repeated dose toxicity studies following oral administration and inhalation with 2-pentanone oxime (2-PO; CAS 623-40-5) available.

Oral

In a combined repeated dose and reproduction / developmental screening test (Key, 2012) according to OECD Guideline 422 and GLP, the test substance 2-PO (CAS 623-40-5) was administered by gavage to Sprague-Dawley rats. 10 males and females per group were treated for two weeks at dose levels of 15, 50 or 150 mg/kg bw/day before pairing. Treatment continued to a total of at least 5 weeks. A control group of 10 male and 10 female rats received the vehicle corn oil at the same volume-dose throughout the same period. Males were killed after at least 5 weeks of treatment and females were killed on Day 7 of lactation. Recovery, over 14 days without treatment, was assessed in five of the control and five of the high dose males and in an extra five unmated females in the same groups, which were treated for 6 weeks before start of recovery. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, gestation length and parturition observations, haematology, blood chemistry, organ weight, pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio and bodyweight of all offspring were assessed. No clinical signs and no mortality occurred during the study. Food consumption was not affected by treatment with the test substance. Males and females receiving 50 and 150 mg/kg bw/day showed low haematocrit (-12 and -17% (m); -9.6% and -11% (f)) and haemoglobin levels (-15.2% and -22% (m); -12% and -16.7% (f)), low red blood cell counts (-15.5% and -31.5% (m);-12.1% and -29.2% (f)), high reticulocytes (+184% and +383% (m); +159% and +346% (f)) and low mean cell haemoglobin concentrations (-3.6% and -6.4% (m); -2.7% and -6.3% (f)) compared to control, and for males only at those dose levels high mean cell volumes (+4.5% and +21%). The high mean cell volume in females was restricted to those receiving 150 mg/kg bw/day (+26.5%). High mean cell haemoglobin levels (+13.2% (m); +18.1% (f)) and high platelet levels (+40.8% (m) and +23.3% (f)) in animals receiving 150 mg/kg bw/day were also evident. After the 14-day off dose period many of the parameters noted to be different to control during treatment had shown complete recovery. Those changes which had not completely resolved included high mean cell haemoglobin level (+11.2%), low mean cell haemoglobin concentration (-2.8%) and high mean cell volumes (+13.5%) for males previously receiving 150 mg/kg bw/day, low red blood cell counts (-7.8%), high mean cell haemoglobin level (+12.2%) and high mean cell volume (+13.4%) for females previously receiving the same dose. The findings in clinical chemistry were similar to control following two weeks of recovery. Enlarged spleen was seen in all males and females treated with 150 mg/kg bw/day and in three out of 10 males treated at 50 mg/kg bw/day. Dark coloration of the spleen was also seen in all males and females treated at 150 mg/kg bw/day, in nine out of 10 males and females treated at 50 mg/kg bw/day and in one out of 10 males treated at 15 mg/kg bw/day. Adjusted mean spleen weight was higher than control in males and females receiving 50 or 150 mg/kg bw/day with a dose response evident. In females receiving 150 mg/kg bw/day there was a slight increase in adjusted mean heart weight. After 14 days recovery, the spleen weights were still slightly higher than control values for males and females, which had received 150 mg/kg bw/day, although no statistical significance was attained for females, and the values were lower than the main study animals. Dark coloration of the kidneys (left and right) was seen in nine females treated with 150 mg/kg bw/day and in three females treated with 50 mg/kg bw/day. After the recovery period, dark coloration of the spleen was seen in three males and two females treated with 150 mg/kg bw/day. At the histopathological examination, haemosiderosis (≥ 15 mg/kg bw/day), congestion (≥ 50 mg/kg bw/day) and extramedullary hematopoiesis (at 150 mg/kg bw/day) in the spleen of both sexes and extramedullary hematopoiesis at 150 mg/kg bw/day in the liver of both sexes were observed. After recovery period, the effects in the liver disappeared. However, in the spleen an increase in the severity of haemosiderosis was observed in males and females previously treated at 150 mg/kg bw/day and a decrease in severity of congestion was also observed in males previously treated at 150 mg/kg bw/day.

Based on the results of the conducted study, a dose level of 15 mg/kg bw/day can be regarded as LOAEL for general toxicity since haemosiderosis, which is regarded as an adverse effect related to haemolytic anaemia, was already observed in the lowest dose group of 15 mg/kg bw/day. At the two higher dose groups multiple effects related to haemolytic anaemia developed, showing the dose-dependent progress of effects related to haemolytic anaemia. Thus, no NOAEL could be determined within this study.

For the results on reproduction and developmental toxicity please refer to the technical dossier chapter 7.8 Toxicity to reproduction.

Inhalation

Sub-acute: 14 days

A sub-acute inhalation toxicity study (Key, 2014) was performed according to OECD Guideline 412 and GLP. The aim of this study was to examine the sub-acute (14-day) inhalation toxicity of 2-PO (CAS 623-40-5) in rats. Four main groups of male and female Sprague Dawley rats (5/sex/group) were exposed nose-only to concentrations (overall mean ± standard deviation) of 52.9 (± 2.8), 149.3 (± 1.9) and 298.9 (± 3.1) ppm for 6 h/day, 5 days/week, during 2 consecutive weeks. The highest target concentration was selected just below the maximum saturated vapour concentration. The study included a concurrent control group of the same size, which was exposed to humidified air only.

Exposure to the test substance up to 298.9 ppm (mean actual concentration measured by total carbon analysis) was welltoleratedas evidenced by the absence of clinical signs of toxicity or adverse effects on growth, food consumption, routinehaematologyand clinical chemistry values, and pathology findings. All animals survived until scheduled sacrifice. Higher spleen weights (absolute and relative to body weight) in males of the mid-and high-dose groups compared to control group animals (absolute: +13.2% and +17.2%; relative: +17.2% and 17.2%), and higher weight of the ovaries (absolute and relative to body weight) in females of the mid-dose group compared to control group animals, were observed. The higher weight of the ovaries was not ascribed to treatment because there was no concentration-related response. The increase in absolute and relative spleen weights of male animals, starting at the mid dose group, were regarded as adverse, taken also 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). Therefore, 149.3 ppm (corresponding to 616.7 mg/m³), was determined to be the No-Observed-Adverse-Effect Concentration (NOAEC) for local and systemic toxicity.Based on the measurement of 2-PO and its metabolites in the blood of the rats after a single (6-h) exposure by inhalation within the sub-chronic inhalation toxicity study it is concluded that 2-PO has reached the blood, and therefore the systemic exposure has occurred at the highest attainable exposure condition.Please, refer to the technical dossier, section 7.1 Toxicokinetics for more details.

Sub-chronic: 90 days

In a sub-chronic inhalation toxicity study (Key, 2016) according to OECD Guideline 413 and GLP 10 male and 10 female rats each were exposed by nose-only inhalation exposure to 0 (control), 49.7 (± 0.7), 149 (± 4) or 301 (± 15) ppm 2-PO (CAS 623-40-5) for 6 h/day, 5 days/week over a 13-week period (65 exposure days). Animals of the main groups were sacrificed on the day after the last exposure. To assess recovery, persistence and/or delayed occurrence of toxicity, two groups of 10 male and 10 female animals each were exposed together with the animals of the control and high concentration groups, and were sacrificed after a 4-week recovery period following the exposure period. The exposure to the test substance was well tolerated by the animals. No treatment-related clinical or ophthalmoscopic abnormalities were observed, and growth and food consumption were not adversely affected by the exposure. Clinical observations did not indicate any narcotic effects in response to the exposure to the test substance. Necropsy did not reveal any treatment-related gross pathological changes. Analysis of blood samples taken in the final stage of the exposure phase from animals of the high concentration recovery group indicated that the test substance reached the blood upon inhalation exposure; blood levels of 2-PO (CAS 623-40-5) and its metabolite methyl propyl ketone (MPK) increased during the course of a 6-h exposure day. After the end of exposure, concentrations of both compounds rapidly decreased and were no longer detectable 6 h after exposure, indicating rapid clearance from the blood. It is concluded that repeated inhalation exposure under these conditions did not result in accumulation of 2-PO in the system(please refer to the technical dossier, section 7.1 Toxicokinetics for more details). Exposure to the test substance resulted in slight changes in clinical pathology parameters in animals sacrificed at the end of the exposure period, e.g. decreased haemoglobin concentration in the high-dosed males (-4.4%), increased percentage of reticulocytes in blood of males in the mid- and high-dose group (+32.5% and +28.9%) and increased plasma bilirubin levels in males and females of the highest dose level (+43.8% and +38.8%). 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 (+18.5 and +27.9%), increased prothrombin time in females (+5.3% and +8%)) 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.

Justification for classification or non-classification

The available data on repeated dose toxicity following oral administration meet the criteria for classification according to Regulation (EC) No 1272/2008 and therefore the test substance will be classified as STOT RE 2 (H373) with the blood and spleen as the target organs.