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Diss Factsheets

Administrative data

Description of key information

Inhalation

- OECD 422, GLP: NOAEC local = 125 mg/m³ (35ppm), NOAEC systemic = 500mg/m³ (140ppm) (BASF, 2019)

- RA 598 -56 -1, OECD 412 (28 -day): NOAEC local = 10 ppm, NOAEC systemic = 50 ppm (TNO, 2019) (highest dose: 250ppm)

- RA 598 -56 -1, OECD 413 (90 -day): NOAEC local = 10.3 ppm, NOAEC systemic = 106 ppm (highest dose tested) (Covance, 2020)

Key value for chemical safety assessment

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Remarks:
combined repeated dose and reproduction/developmental tox screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 2018 - Aug 2019
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)
Version / remarks:
Jul 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Sponsor: BASF SE, Ludwigshafen, Germany
- Purity: 99.9%
- Storage conditions: Room temperature
- Homogeneity: Given
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Charles River Laboratories, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation:males (10-11 weeks old) and females (9 weeks old)
- Weight at study initiation: (P) Males: 380 g; Females: 218 g;
- Housing:
From delivery until randomization, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECNIPLAST, Hohenpeißenberg, Germany. From randomization onwards, the rats were housed individually in Polycarbonate cages type IIIsupplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel,Germany, with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
• Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
• During exposure, male and female rats (until gestation) were housed in wire cages, type. DK III supplied by Becker & Co., Castrop-Rauxel, Germany, and female rats (from PND4 onwards) in perforated polycarbonate cages type III. “Exposure systems”.Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ Lignocel® block large, J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany) were added. In addition, in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX B.V., Elst, Netherland were added.

- Diet: ad libitum; Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%):45-65%
- Air changes (per hr):15 times per hour.
- Photoperiod (hrs dark / hrs light):12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole-body exposure systems
- Method of holding animals in test chamber: The animals were kept single in wire cages located in a glass-steel inhalation chamber.
- Source and rate of air: 1.1 m³
- Method of conditioning air: The animals were housed in fully air-conditioned rooms in which central air conditioning
- Temperature, humidity in air chamber: temperature of ~22°C and relative humidity of ~50%.
- System of generating particulates/aerosols:
• Piston metering pumps KP 2000 (DESAGA; SARSTED AG & Co, Nürnbrecht, Germany)
• Atomization vaporizers (glass) with thermostat (BASF SE, Ludwigshafen, Germany)
• Thermostat (JULABO Labortechnik GmbH, Seelbach, Germany)
The test substance was used unchanged. For each concentration, the test substance was supplied to the two-component atomizer of a thermostated vaporizer at a constant rate by means of the metering pump. The vapour/ air mixture was generated by spraying the substance with compressed air into a counter current of conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 4°C). Thereafter it was further mixed with conditioned supply air and passed into the inhalation system.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Calculation of nominal concentrations
The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations.

Analytical determination of concentrations
Principle: The concentrations of the test substances in the atmospheres were determined by calibrated on-line gas chromatography method (GC) using on-line microGC.
To calibrate the microGC, an appropriate amount of test substance were weighed into gas sampling tubes with defined volume. The gas sampling tube is tempered that the test substance is evaporated completely within the tube. The so prepared atmospheres are sampled by microGC. A calibration line was formed by the pair of substance concentration and peak area. The method authorized by the contributing scientist described in detail the calibration process, the calibration curve as well as accuracy and reproducibility of the method. The non-GLP raw data of methods description were archived with the study raw data.
During the study, atmospheres were sampled by microGC via a stream selector, which switch through all concentration groups including the control group. Between the measurement of each group, an appropriate flush time was included to avoid carry over effects. The measured data were transferred to BaseLab, which generated hourly means for each test group. Daily means and standard deviations were calculated based on hourly means of each concentration for each exposure.
Duration of treatment / exposure:
Groups of 10 male and 10 female Wistar rats (F0 animals) per test group were exposed in a whole-body inhalation system to dynamic atmosphere of the test substance for 6 hours per day on each day.The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 6 days postmating in males, and the gestation (up to GD 19) and the lactation period in females from PND 4 onwards up to the day of scheduled sacrifice of the animals. The target concentrations were 125 mg/m³ (34 ppm1), 500 mg/m³ and 1500 mg/m³). A concurrent control group was exposed to conditioned air. For adaptation to the exposure conditions the animals were placed into exposure cages before start of the exposure period (pre-exposure period) on study days -5, -4 and -1 (details are available in the raw data). This pre-exposure was performed in the animal room.

Duration of the exposure:
Males:
a) 14 days premating
b) up to 14 days mating
c) sacrifice after a minimum of 28 days after the first exposure
Females:
a) 14 days premating
b) up to 14 days mating
c) during the pregnancy up to and including GD 19
d) after lactation from PND 4 onwards until one day before necropsy

The animals did not have access to water or feed during the exposure.

For adaptation to the exposure conditions the animals were placed into exposure cages before start of the exposure period (pre-exposure period) on study days -5, -4 and -1. This pre-exposure was performed in the animal room.
Frequency of treatment:
6 hours/ day, every day
Dose / conc.:
125 mg/m³ air (nominal)
Remarks:
123 mg/m³ analytical
Dose / conc.:
500 mg/m³ air (nominal)
Remarks:
505 mg/m³ analytical
Dose / conc.:
1 500 mg/m³ air (nominal)
Remarks:
1513 mg/m³ analytical
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
The animals were delivered and subjected immediately to the acclimatization period in which they were adapted to the surroundings.
Prior to the pre-exposure period, the animals were distributed according to body weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed ± 20 percent of the mean weight of each sex. The list of randomization instructions was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists were created. The list of randomization instructions was compiled with a computer (Laboratory data processing, Experimental Toxicology and Ecology, BASF SE).
All animals were exposed to the respective concentrations of test substance for 6 hours a day according to the time schedule (exception: no exposure on the day of FOB/MA). Control animals were exposed to conditioned air. All animals were observed daily for any clinical signs during the study period.
A detailed clinical observation (DCO) was performed in all animals once before the first exposure and weekly thereafter.
Males and females from the same test group were mated, after two weeks of treatment, overnight at a ratio of 1:1.
The females were allowed to deliver and rear their pups until PND 4 (standardization) or PND 13.
After the standardization of the litters, the parental female animals were exposed to the respective concentrations of test substance for 6 hours a day until one day before scheduled sacrifice.
Blood samples were taken from surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement.
On study day 30, a functional observational battery and motor activity measurement were carried out in five male animals per group.
On study day 57, a functional observational battery and motor activity measurement were carried out in five female animals (with litter) per group.
Blood samples were taken from all F0 parental male animals of all test groups shortly before sacrifice and from all F0 parental female animals of all test groups on PND 14.
The male and female animals were sacrificed 34 and 59 days, respectively, after the beginning of the treatment, and examined.

Standardization of litters (F1 generation pups)
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Surplus pups were sacrificed. Standardization of litters was not performed in litters ≤ 8 pups.

Pups after standardization
On PND 4, as a result of standardization, the surplus pups or 2 preferably female pups per litter, respectively, were sacrificed under isoflurane anesthesia by decapitation. Blood were sampled for determination of thyroid hormone concentrations. All pups were examined externally and eviscerated; their organs were assessed macroscopically.

Pairing of F0 generation parental animals
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 6.30 - 9.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1".

Observations and examinations performed and frequency:
CLINICAL EXAMINATIONS
Parental animals
Mortality:
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the BASF SE Laboratory for Pathology, Experimental Toxicology and Ecology, Ludwigshafen, Germany.
Clinical observations:
The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days. During exposure, a group wise examination only was possible. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.
Food consumption:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14, and 14-20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10 and 10-13.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.
Body weight data:
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 10 and 13.
Females without litter or waiting for necropsy, were weighed weekly. The body weight data of these individuals are not reported in the Summary but in the Individual Tables (PART II).
Detailed clinical observations:
Detailed clinical observations (DCO) were performed in all animals once prior to the first treatment (day 0) and at weekly intervals during the exposure period. The examinations startedin the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed: Abnormal behavior in “handling”, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos (Protruding eyeball), Assessment of the feces excreted during the examination (appearance/consistency), Assessment of the urine excreted during the examination, Pupil size
Functional observation battery:
A functional observation battery (FOB) was performed in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. There is no exposure of the concerning animals as well as the other 5 animals of the same test group. At least one hour before the start of the FOB the animals were transferred singly to Polycarbonate cages (floor area about 800 cm²). The cages were placed in the racks in a randomized order (randomization based upon animal’s number). Drinking water was provided ad libitum whereas no food was offered during the measurements. The FOB started with passive observations, without disturbing the animals, followed by removal from home cage, and open field observations in a standard arena. Thereafter, sensorimotor tests and reflex tests were conducted. The examinations were carried out by trained technicians which performed positive control studies as part of their training. Another technician documented all findings and values obtained. The findings were ranked according to the degree of severity, if applicable.
Home cage observations:
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings
Open field observations:
The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and observed for at least 2 minutes. Following parameters were examined: Behavior on removal from the cage, Fur, Skin, Salivation, Nasal dischare, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/ stereotypies, Gait, Activity/arousal level, Feces (appearance/consistency) within 2 minutes, Urine (amount/color) within 2 minutes, Rearings within 2 minutes, Other findings
Sensory motor tests/reflexes:
The animals were removed from the open field and subjected to following sensorimotor or reflex tests: Reaction to an object being moved towards the face (Approach response), Touch sensitivity (Touch response, Vision (visual placing response), Pupillary reflex, Pinna reflex, Audition (Startle response), Coordination of movements (Righting response), Behavior during handling, Vocalization, Pain perception (Tail pinch), Other findings, Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test
Motor activity measurement:
The Measurement of motor activity (MA) was measured at the end of the exposure period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.
Estrous cycle determinations:
For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization (the estrous cycle data of these individuals were not be reported and can be found in the raw data). For a minimum of 2 weeks prior to mating estrous cyclellength was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
Male reproduction data:
The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs. For the males, mating and fertility indices were calculated for F1 litters according the formulas given in OECD TG 422 (2016).
Female reproduction and delivery data:
The pairing partners, the number of mating days until vaginal sperm was detected and gestational status was recorded for F0 females. For the females, mating, fertility and gestation indices were calculated for F1 litters according to the formulas given in OECD TG 422 (2016).
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula (given in OECD TG 422 (2016)): Live birth index (%) = (number of liveborn pups at birth / total number of pups born) * 100.
The implantations were counted and the postimplantation loss (in %) was calculated according to the following formula (given in OECD TG 422 (2016)): Postimplantation loss (%) = ((number of implantations - number of pups delivered) / number of implantations) * 100.

CLINICAL PATHOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The parameters listed below were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
Hematology:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), differential blood count, Reticulocytes (RET):
Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Only evaluated blood smears were archived.
Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Measured parameter was prothrombin time.

Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the following clinicochemical parameters: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Na, K, Cl, inorganic Phosphate (INP), Ca, Urea, Creatinine, Glucose, total Bilirubin, total protein, Albumin, Globulins, Triglycerides, Cholesterol, Bile acids.
Thyroid hormones
Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling. All generated serum samples were frozen at -80°C until measurement.
Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH). (For technical reasons the values of PND 13 pups (males Nos. 201-240, females Nos. 301-340) were listed in the mean and individual tables under test groups 10, 11, 12 and 13 instead of 0,1, 2 and 3). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
Sacrifice and pathology:
Necropsy:
All parental animals were sacrificed under pentobarbitone anesthesia. The left and right brachial vessels were opened, by deep cuts through the pectoral muscles along both sides of the rib cage. Caution was exercised to avoid destruction of the axillary lymph nodes. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
Organ weights
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (terminal body weight), Epididymides, Ovaries, Prostate (ventral and dorsolateral part together, fixed), Seminal vesicles with coagulating glands (fixed), Testes, Thyroid glands (with parathyroid glands) (fixed), Uterus (with cervix)
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands (fixed), Brain, Heart, Kidneys, Liver, Lungs, Spleen, Thymus (fixed).
All paired organs were weighed together (left and right).

Organ/tissue fixation
The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Esophagus, Epididymides (modified Davidson’s solution), Extraorbital lacrimal glands, Eyes with optic nerve (modified Davidson’s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E, 1964), Vagina.
Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings. For further details on histopathological examination please see attached background material.
Special attention was given to the stages of spermatogenesis and histopathology of interstitial testicular cell structure.
The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
A correlation between gross lesions and histopathological findings was attempted.
Whenever in the ovary the diagnosis: „no abnormalities detected” was used that implies that all different stages of functional bodies (especially corpora lutea) were present and normal.
Peer review
After completion of the histopathological assessment by the study pathologist an internal peer review was performed including prostate glands and ovaries of all animals of all test groups. Results presented in this report reflect the consensus opinion of the study pathologist and the peer review pathologist.
Statistics:
Food consumption, body weight (bw) and bw change (parental animals and pups; for pup weights, litter means were used), gestation days, anogenital distance, anogenital index: Simultaneous comparison of all dose groups with control group (CoG) using DUNNETT test (2sided)
Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups: Pair-wise comparison of each dose group with CoG using FISHER'S EXACT test (onesided)
Mating days until day 0 p.c., %postimplantation loss, pups stillborn, %perinatal loss, nipple development, Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival index:Pair-wise comparison of the dose group with CoG using WILCOXON test (onesided) with BONFERRONI-HOLM adjustment
%live male day x, %live female day x: WILCOXON test (2sided)
Number of cycles and Cycle Length, Rearing, grip strength of fore limbs and hind limbs, landing foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of the dose groups with CoG was performed using WILCOXON test (2sided) for the equal medians.
Blood parameters: Parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with CoG was performed using WILCOXON-test (2sided). For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided).
Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with CoG was performed using WILCOXON-test (2sided)for the equal medians.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During premating and mating periods, all parental male and female animals (10 out of 10) of test group 3 (1500 mg/m³) showed salivation, piloerection and reduced attention during exposure. Furthermore, a discolored fur (reddish) occurred in one female of test group 3 during premating and in each two males and females of test group 3 during mating. Salivation, piloerection and reduced attention lasted up to scheduled sacrifice (during postmating period) in all male animals of test group 3. These clinical signs were also seen in all test group 3 female animals during gestation period. Furthermore, a discolored fur (reddish) occurred in six females of test group 3 during gestation. During lactation period, all female animals of test group 3 had piloerection and their attention was reduced during exposure.
The above-mentioned clinical findings in test group 3 male and female animals were assessed as treatment-related and adverse.
No clinical signs or changes of general behavior which may be attributed to the test substance, were detected in any male or female F0 parental animals at concentrations of 125 or 500 mg/m³ during premating, mating, gestation, lactation and postmating periods. For one female animal of test group 2 (No. 127 - 500 mg/m³) hypothermia and piloerection were recorded at the end of the gestation period (GD 23-24). In the litter of this female, all pups were stillborn (PND 0). Furthermore, test group 3 female animal No. 133 (1500 mg/m³) had an opacity on its right eye during PND 15-18. These single events were assessed to be incidental and not related to treatment.
One sperm-positive female of the control group (No. 104) did not deliver F1 pups.

Detailed clinical observations (DCO)
No test substance-related findings were observed in male and female animals of all test groups (125, 500 or 1500 mg/m³).
One spontaneous finding occurred in test group 3 during DCO on day 56, i.e. an opacity on the right eye of female animal No. 133.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During premating period, test group 3 F0 male animals (1500 mg/m³) lost weight at all time points. Thus, the mean body weight (change) was statistically significantly reduced during thewhole premating period. During premating, mating and postmating, their mean body weight was constantly decreased (up to -9%) compared to the concurrent control.
The mean body weights of the test group 3 F0 parental female animals (1500 mg/m³) were statistically significantly decreased compared to the concurrent control values during gestation (GD 7-20: up to -9%) and lactation (PND 7-13: up to -12%). In test group 3, the mean body weight change of the F0 females was statistically significantly reduced during premating (0-13) showing a body weight loss, during gestation (GD 0-20: -21% below control) and lactation (PND 1-13: -52% in comparison to the concurrent control). The body weight decrease of the test group 3 male and female parental animals (1500 mg/m³) was assessed as treatment-related and adverse.
In test group 2 (500 mg/m³), F0 parental males gained less weight during premating (attaining statistical significance) but recovered afterwards during mating. Since the body weight gain was not affected during mating and postmating, this marginal decrease during one short time period was not assessed as treatment-related and adverse.
The mean body weight (change) of the test group 1 males and females and test group 2 females were comparable to the concurrent control group during the entire study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the F0 parental males of test group 3 (1500 mg/m³) was statistically significantly reduced during premating period(up to 24% below concurrent control). If calculated for the whole premating period (days 0-13), these males consumed approx. 23% less food than the controls.
Consistently to the males, food consumption of the F0 parental test group 3 females (1500 mg/m³) was statistically significantly reduced during premating (up to -20%; days 0-13: approx. 17% below control), gestation (GD 0-20: -13%) and lactation (PND 4-13: up to -23%; PND 1-13: approx. 17% below control).
The reduction in food consumption of the test group 3 males and females in all treatment periods during lactation was assessed as treatment-related and adverse.
In test group 2 (500 mg/m³), the food consumption of thetest group 2 females was generally comparable to control during premating and gestation. During lactation, food consumption of the F0 females was reduced compared to control from PND 4 onwards (PND 4-7: -10%, without statistical significance; PND 7-13: -14%, with statistical significance).
For the test group 2 females, the reduction in food consumption during lactation was assessed as treatment-related but not as adverse since it caused neither a corresponding reduction of body weight/body weight gain nor any other measurable effect on the well-being of those animals.
Food consumption of the F0 male and female animals in test group 1 (125 mg/m³) and of the F0 males in test group 2 (500 mg/m³) was generally comparable to the respective control group during the entire treatment period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
At the end of the administration period, in parental males of test group 1 (125 mg/m3) relative neutrophil counts were significantly decreased, but this alteration was not concentration dependent In dams at PND14, absolute reticulocyte counts were significantly lower compared to study controls. However, this change was also not concentration-dependent. Therefore, these alterations were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in parental males and dams at PND14 of test group 3 (1500 mg/m3) bilirubin was slightly increased above historical controls values in both sexes, but attained statistical significance in females only. Stress as well as fasting lead to an increase in serum bilirubin. Food consumption (and body weight) of high dose animals was severely reduced, adrenal gland weights were reduced as a result of stress (all likely caused by the local corrosive effects in the nose and larynx). Consequently, the slight change in bilirubin is not considered to be a direct effect of treatment, but secondary to stress and reduced food consumption.
Values for bile acids were also slightly above historical control values, though differences were not statistically significant. In females, no dose response was observed as the highest value occured in low dose animals. For males, only 2 of 5 animals had serum bile values just above the historical control data (though several values in the same range occured in the historical controls, but were marked as outliers). Because of the small increase, no statistical significance and no dose repsonse in females, the change was not considered to be related to treatment.
In parental males of test group 3 (1500 mg/m3) alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were significantly increased. In males of test groups 2 and 3(500 and 1500 mg/m3) chloride levels were significantly decreased whereas in males of test groups 1, 2 and 3 (125, 500 and 1500 mg/m3) inorganic phosphate levels wer esignificantly higher compared to study controls. However, all mentioned parameter values were within historical control ranges (males, ALT 0.55-0.90 μkat/L; AST 1.31-2.11 μkat/L; chloride 97.9-104.1 mmol/L; inorganic phosphate 1.45-2.06 mmol/L). Therefore, the mentioned alterations were regarded as incidental and not treatment-related.
In females of test group 3 (1500 mg/m3) potassium levels were significantly increased. The mean was above the historical control range (females, potassium 4.23-4.90 mmol/L). This was the only changed electrolyte among these individuals. Therefore, this change is regarded as treatment-related, but non-adverse (ECETOC Technical Report No 85, 2002).
In parental males (test groups 1, 2 and 3; 125, 500 and 1500 mg/m3) and in male and female pups at PND13 (test groups 11, 12 and 13; 125, 500 and 1500 mg/m3), no treatment-related alterations of T4 and TSH levels were observed.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observational battery:
No test substance-related findings were observed in male and female animals of all test groups during the home cage observation.
One male of the mid-concentration group (No. 25 - 500 mg/m³) showed manege movements during the home cage observation. Since only one animal showed this finding, it was not assessed as treatment-related and adverse.

Open field observations:
One male of the high concentration group, two males, each, of the mid- and low-concentration groups and one male of the control showed slight resistance against handling. Furthermore, two females of the mid-concentration and one female of the low-concentration group showed the same finding during open field observation. Since there was no relation to concentration, it was assessed as spontaneous. The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.

Sensorimotor tests/reflexes:
One male animal, each, of test groups 1 and 3 showed very frequent vocalizations when touched. One test group 3 male had a weak or retarded reaction to the stimulus during the pain perception test. These findingswere not assessed as treatment-related and adverse since they occurred only in individual animals and all males showed neither clinical signs nor changes in associated parameters in this study.
There were no testsubstance-related findings in male and female animals of all test groups.

Motor activity measurement:
No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all concentration groups in comparison to the concurrent control group. The animals showed a normal habituation to the test environment. Motor activity measurement (single value) was statistically significantly above the concurrent control value in males of test group 3 (1500 mg/m³) during interval 3 (513.8 vs. 236.4 in control). Since the increase was a single, isolated event, it was assessed as incidental and not treatment related.

Quantitative Parameters:
No test substance-related impaired parameters were observed in male and female animals of all test groups. The statistically significantly increased number of rearing in the females of test group 1 was assessed as incidental since there was no relation to concentration.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The significantly increased absolute and relative weights of adrenal glands in females of test group 3 were regarded as treatment related, but non-adverse. Absolute adrenal gland weights were at the upper end of historical control values whereas relative adrenal gland weights were slightly above the range of historical controls. Since the animals showed a significantly reduced terminal body weight, the finding was regarded as a secondary, stress-related response without any histopathological correlate.
The significantly decreased absolute heart weight in males and females of test group 3 as well as the significantly increased relative weights of brain, epididymides and testes in males of test group 3 were related to the significantly decreased terminal body weight in these animals.
The significantly decreased absolute weights of prostate glands in test groups 2 and 3 males, the significantly decreased weights of seminal vesicles (absolute: test groups 1 and 2, relative: test group 2,) the significantly increased absolute and relative uterus weights in test group 2 females and the significantly increased liver weights in males of test groups 1 and 2 were regarded to be incidental, as there was no concentration-response-relationship and no histopathological findings, that would explain the organ weight changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in the nasal cavity (all four levels) of test groups 2 and 3 males and females, in the larynx (level I) of test group 3 males and females.

The nasal cavity (level I-IV) showed a degeneration/regeneration of the olfactory epithelium, characterized by one or more of the following findings: increased intercellular spaces, irregular epithelial architecture, necrotic epithelium, and an increased nuclear/ cytoplasmic ratio. Multifocally, a formation of glandular-like structures within the olfactory epithelial layer was observed. The degeneration/regeneration was mainly located in the dorsal part of the nasal septum and in the ethmoid turbinate. The severity varied from minimal to marked and was concentration-related increased.

Almost all male and female animals of test group 3 showed a minimal (grade 1) to marked (grade 4) degeneration/regeneration of the olfactory epithelium at all four levels. Few male and female animals of test group 2 showed a minimal degeneration/regeneration of the olfactory epithelium in levels II and III of the nasal cavity. The degeneration/regeneration of the olfactory epithelium was regarded as treatment-related.
Level I of the larynx showed an increased incidence of minimal epithelial alteration in males and females of test group 3. The increased incidence of epithelial alteration was regarded as treatment-related.

In the prostate of test group 3 males, a slightly increased incidence and severity of inflammation was observed. The inflammation was mainly located in the ventral part of the prostate gland and did not differ morphologically from the inflammation occurring in the control animal. Prostatic inflammation is a common background finding in rats (Creasy et al., 2012) and the incidence of this finding in the current study was rather low despite the slight increase in test group 3 animals. Therefore, and since there were no additional pathologic findings at the male reproductive tract nor any hints regarding a reduced fertility, a relation to treatment was regarded as unlikely. The slightly increased incidence of eosinophilic droplets in the kidneys in male animals of test group 3 (4/5 in test group 3 vs. 2/5 in control) was regarded as incidental, since eosinophilic droplets belong to the very common background findings in male rats (McInnes, 2012), that reach up to 100% in the historical controls. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The stages of spermatogenesis in the testes of males of the test group 3 were comparable to those of the controls. In test group 3 females the different stages of functional bodies in the ovaries were present and comparable to the control animals.

Fertility
The female animal (No. 104), which was not pregnant, did not show relevant histopathological findings of the reproductive organs. All other organs were not examined histopathologically. The male mating partner (No. 4) did not show relevant histopathological findings.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No treatment-related alterations of T4 and TSH levels were observed.
Key result
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
125 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
500 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Critical effects observed:
no
Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEC (no observed adverse effect concentration) for local toxicity of Dimethyl(propyl)amine in the respiratory tract was the lowest tested concentration of 125 mg/m³ based on adverse effects such as degeneration/regeneration of the olfactory epithelium in the nasal cavity of the parental animals after inhalation (vapor) exposure to 500 and 1500 mg/m³.
The NOAEC for systemic toxicity was the mid concentration of 500 mg/m³ as adverse clinical signs, a reduction in food consumption together with a decrease in body weight (change) occurred in both sexes at the highest concentration of 1500 mg/m³.
Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
Range finder
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Dec 2017 - Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
7-day DRF to select the concentration levels for the subacute main study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Arkema, La Chambre, France
- Batch No.of test material: SAMP160321
- Expiration date: 20 Nov 2018
- Purity: 99.41%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature (in a dry, cool and well-ventilated place, storage in hermetically closed bottle at +4 °C)
- Stability under test conditions: not specified
- Solubility and stability of the test substance in water: completely soluble
- Reactivity of the test substance with water: exothermic reaction

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
- other information:
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
Rats were chosen, because this species is normally used in toxicity studies and is accepted by the relevant regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 251 g (males), 166 g (females)
- Fasting period: no
- Housing: single (during exposure), goup of five, seperated by sex (non-exposure)
- Diet: ad libitum
- Water: ad libitum:
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY:
- Food: Analyzed for nutrients and contaminants, including certificate
- Water: Checked routinely (physical, chemical, and microbial examination), including results

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 Jan 2018 (first exposure, day 0) To: 17 Jan 2018 (day 7)
Route of administration:
inhalation: mist
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: inhalation chambers consisting of a cylindrical aluminium column, surrounded by a transparent cylinder (a modification of the chamber made by ADG Developments Ltd., Codicote, Hitchin, Her ts, SG4 8UB, United Kingdom)
- Method of holding animals in test chamber: animals were secured in plastic animal holders (Battelle)
- System of generating particulates/aerosols: To generate the test atmospheres, a continuous flow of liquid test material3, controlled by a peristaltic pump (Minipulse 3, Gilson, Velliers le Bel, France) was allowed to evaporate in a mass flow controlled (Bronkhorst Hi Tec, Ruurlo, the Netherlands) stream of compressed dry air, by directing it through a glass evaporator which was kept at a constant temperature of 48˚C by circulating heated water.
- Temperature, humidity, pressure in air chamber: 22 +/- 3 °C, 30-70%, positive pressure in the central column and a slightly negative pressure in the outer cylinder
- Air flow rate: 1 liter/min

TEST ATMOSPHERE
- Brief description of analytical method used: Total carbon analysis
- Samples taken from breathing zone: yes. Empty ports were used for test atmosphere sampling
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the first exposure, calibration was performed.
10 ppm: On 21 and 22 December 2017, concentrations of 7.8, 8.5, 9.2, 9.7, 9.8, 9.8, 9.9, 10.9, 11.5, 19.7 and 21.5 ppm were analyzed
100 ppm: On 20 December 2017, concentrations of 73, 79, 109, 111, 124 and 127 ppm were analyzed
500 ppm: On 9 January 2018, concentrations of 358, 374, 440, 474, 615 and 727 ppm were analyzed
1000 ppm: On 19 December 2017, concentrations of 852, 1005, 1079, 1142, 1281 and 1338 ppm were analyzed
Duration of treatment / exposure:
7 d (5 exposure days in total)
Frequency of treatment:
6 h/d, 5 d/w
Dose / conc.:
10 ppm (nominal)
Remarks:
10.0 (± 0.1) ppm (analytical)
Dose / conc.:
100 ppm (nominal)
Remarks:
100.5 (± 0.9) ppm (analytical)
Dose / conc.:
500 ppm (nominal)
Remarks:
497.2 (± 6.5) ppm (analytical)
Dose / conc.:
1 000 ppm (nominal)
Remarks:
998.1 (± 4.3) ppm (analytical)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Selection of target concentrations for the concentration range finding study were based on the 4h LC50 that was found to be ca. 3400 ppm; no mortality was observed at a lower concentration of 2300 ppm. Although no repeated-dose inhalation toxicity data were available for Dimethylethylamine, there were data for the toxicity of the comparable substance Trimethylamine (CAS no. 75-50-3). The 4h LC50 was comparable at ca. 2400-3500 ppm. In a subacute study, mild toxicity was observed at the low concentration of 75 ppm, while mild toxicity was observed at a concentration as low as 10 ppm in a chronic study (all information available at http://www.echa.eu); thus, to enable identification of a NOAEC and characterize the concentration-response relationship, a broad concentration range was chosen for this range finding study.
Positive control:
not included
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily in the morning, halfway through exposure, and afternoon

DETAILED CLINICAL OBSERVATIONS: Yes, if necessary
- Time schedule: daily, afternoon

BODY WEIGHT: Yes
- Time schedule for examinations: 2 days before start of exposure, prior to first exposure (day 0), on day 4, and on scheduled sacrifice

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Time schedule: Once weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, adrenals, heart, kidneys, liver, lungs (with trachea), spleen, testes, ovaries.
HISTOPATHOLOGY: Yes, larynx, kidneys, nasal turbinates, tracheobronchial lymph nodes, trachea, lungs
Statistics:
- Ancova/Anova & Dunnett (or Kruskal-Wallis & Dunnett on Ranks): Pretreatment body weight, body weight after initiation of treatment, clinical pathology (hematology, clinical chemistry), bronchoalveolar lavage parameters, organ weights, sperm motility (numerical), sperm count, testicular sperm count
- No statistics were applied (only one cage/sex/group): food consumption
- Kruskal-Wallis & Wilcoxon: Estrus cyclicity: mean length of the longest cycle, number of complete cycles in the test period; sperm motility (expressed as %), sperm morphology
- Chi-Squared & Fisher’s Exact: Incidences of histopathological changes; Estrus cyclicity: number of acyclic females, number of animals with prolonged estrus period
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 ppm: all males and most females displayed dyspnea, sniffing, irregular respiration, hunched posture, ataxia, blepharospasm and piloerection and hypoactivity. Mouth breathing observed in 4/5 males and 1/5 females. Most animals displayed salivation, some grunting, chewing movement and shallow breathing.
500 ppm: dyspnea, shallow breathing, hunched posture and piloerection were noted in about half of males after first exposure
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 ppm: Concentration-related substantial body weight loss in males (16% average loss of body weight between days 0 and 7). In females, statistically significant reduced growth
500 ppm: Reduced growth in males
100 ppm: Slightly reduced body weight growth in males
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1000 ppm: Decreased food intake in males and females
500 ppm: Decreased food intake in males
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
1000 ppm: Relative weights of lungs and heart statistically significantly increased in males and females. In males, relative testes and adrenals weights increased and relative liver weights decreased
500 ppm: In males, relative testes weights increased
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 ppm: Lungs of 4/5 males showed red spots. Small thymus was found in 2/5 males and 2/5 females. A small thymus found in 2/5 males and females
Some single animals in the other treatment groups showed red spots in the lungs that probably were related to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1000 ppm: Level 2 was most affected with the highest score grades for (granulocytic) inflammation, ulceration and necrosis. The damaged areas were mainly the nasal septum, the conchae and the lateral walls of the nasal cavity. In severe cases of necrosis, parts of the conchae and/or nasal septum were completely gone. High score grades for inflammation and ulceration were also observed in levels 3 and 4, while slightly lower grades were scored in levels 5 and 6. The remains of the damaged epithelium showed large areas with squamous metaplasia. In the necrotic areas sometimes colonies of (most likely) opportunistic bacteria were seen. Larynx: Minimal to mild granulocytic inflammation in 8/10, mild squamous metaplasia in 8/10 and minimal to mild ulceration in 4/10 animals. Trachea/bronchi: minimal to mild granulocytic inflammation in 9/10 animals. Lungs: alveolar hemorrhages in 4/10 animals, increased bronchus associated lymphoid tissue in 4/10, granulocytic inflammation of bronchi in 8/10, and a granulomatous inflammation in 2/10 animals.
500 ppm: olfactory epithelial atrophy in most animals. inflammatory changes were seen in several animals, in levels 1 to 4, occasionally accompanied by necrosis or ulceration. Minimal to mild granulocytic inflammation in 4/10 animals. Minimal to mild granulocytic inflammation in 3/10 animals.
100 ppm: Minimal to mild atrophy of the olfactory epithelium in levels 3, 4 and 5, mainly in the dorsal meatus.
Histopathological findings: neoplastic:
not examined
Details on results:
Under the conditions of this 7-day concentration range-finding study, inhalation exposure to the test substance at target concentrations of 10, 100, 500 and 1000 ppm resulted in the following treatment-related changes:
• At 1000 ppm, marked toxicity was observed; in nearly all animals, dyspnea, sniffing, irregular respiration, hunched posture, ataxia, blepharospasm, piloerection and hypoactivity were observed. Body weight of males exposed to the top concentration were 22% lower than controls (on day 7); for females the reduction in body weight was 15%. Relative weight of the heart and lungs was increased in male and female animals. When assessing histopathology, severe olfactory epithelial atrophy was observed in the nasal tissues. Milder effects were observed in the larynx, trachea/bronchi and lungs, mainly of inflammatory nature.
• At 500 ppm, transient clinical abnormalities (dyspnea, shallow breathing, hunched posture and piloerection) were observed in males especially. Minimal to mild atrophy of the nasal olfactory epithelium was observed in most animals when assessing nose tissue levels 3 and 4 (out of the 6 levels assessed).
• At 100 ppm, minimal atrophy of the nasal olfactory epithelium was still observed in most animals, with 2 cases of mild atrophy observed at a single level of the nose in 2 animals.
• At 10 ppm, no exposure-related toxicity was seen. Thus, the No-Observed-Adverse-Effect- Concentration (NOAEC) in this range finding study was placed at 10 ppm (target concentration).
Dose descriptor:
NOAEC
Effect level:
10 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
histopathology: non-neoplastic
Critical effects observed:
no

Table 1: Clinical observations in males

Observation Type: All Types Sex: Male

From Day -2 (Start Date) to 7 (Start Date)

0 ppm

10

ppm

100

ppm

500

ppm

1000

ppm

DEAD Killed scheduled

5

5

5

5

5

RESPIRATION Sniffing

0

0

0

0

5

RESPIRATION Grunting

0

0

0

0

2

RESPIRATION Irregular

0

0

0

0

5

RESPIRATION Dyspnoea

0

0

0

3

5

RESPIRATION Shallow

0

0

0

1

0

RESPIRATION mouth breathing

0

0

0

0

4

MOUTH Salivation

0

0

0

0

3

BEHAVIOUR Hunched posture

0

0

0

3

5

BEHAVIOUR Hypoactive

0

0

0

0

5

BEHAVIOUR Ataxia

0

0

0

0

5

SKIN Piloerection

0

0

0

5

5

EYES Blepharospasm

0

0

0

1

5

Table 2: Clinical observations in females

Observation Type: All Types Sex: Female

From Day -2 (Start Date) to 7 (Start Date)

0 ppm

10

ppm

100

ppm

500

ppm

1000

ppm

DEAD Killed scheduled

5

5

5

5

5

RESPIRATION Sniffing

0

0

0

0

5

RESPIRATION Grunting

0

0

0

0

1

RESPIRATION Irregular

0

0

0

0

4

RESPIRATION Dyspnoea

0

0

0

0

5

RESPIRATION Shallow

0

0

0

0

1

RESPIRATION Sneezing

0

0

0

0

1

RESPIRATION mouth breathing

0

0

0

0

1

MOUTH Salivation

0

0

0

0

3

MOUTH chewing movement

0

0

0

0

2

BEHAVIOUR Hunched posture

0

0

0

0

5

BEHAVIOUR Hypoactive

0

0

0

0

5

BEHAVIOUR Ataxia

0

0

0

0

5

SKIN Piloerection

0

0

0

5

5

EYES Blepharospasm

0

0

0

0

5

Table 3: Body weight, males

Sex: Male

 

Bodywt

day-x(g)

 

[G]

Bodywt

day0(g)

 

[G]

Bodywt

 

(g)

 

[C]

Bodywt

 

(g)

 

[C]

-2

0

4

7

0 ppm

Mean

238.74

250.60

257.22

270.28

 

SD

12.47

12.92

16.19

16.66

 

N

5

5

5

5

10 ppm

Mean

241.14

251.68

255.16

266.16

 

SD

13.73

14.92

14.17

16.11

 

N

5

5

5

5

100 ppm

Mean

240.50

252.62

252.40

260.36

 

SD

16.63

17.44

15.54

16.57

 

N

5

5

5

5

500 ppm

Mean

239.66

248.42

237.30 **

237.44 *

 

SD

13.61

14.77

16.13

18.56

 

N

5

5

5

5

1000 ppm

Mean

241.08

250.74

218.26 **

210.72 **

 

SD

15.37

16.30

11.24

10.57

 

N

5

5

5

5

[G] - Ancova/Anova & Dunnett

[C] - Ancova/Anova & Dunnett {Covariate: Bodywt day 0}: * = p < 0.05; ** = p < 0.01

Table 4: Body weight, females

Sex: Female

 

Bodywt

day-x(g)

 

[G]

Bodywt

day0(g)

 

[G]

Bodywt

 

(g)

 

[C]

Bodywt

 

(g)

 

[C]

-2

0

4

7

0 ppm

Mean

165.04

164.42

168.86

179.60

 

SD

7.88

3.55

3.98

10.01

 

N

5

5

5

5

10 ppm

Mean

166.18

170.86

173.38

178.88

 

SD

8.08

5.82

4.21

6.97

 

N

5

5

5

5

100 ppm

Mean

165.56

167.18

171.60

176.16

 

SD

7.10

12.44

5.72

7.73

 

N

5

5

5

5

500 ppm

Mean

164.02

165.84

167.10

175.18

 

SD

9.77

9.40

9.52

9.71

 

N

5

5

5

5

1000 ppm

Mean

162.96

162.82

154.72 **

151.94 **

 

SD

8.33

8.84

8.40

9.01

 

N

5

5

5

5

[G] - Ancova/Anova & Dunnett

[C] - Ancova/Anova & Dunnett {Covariate: Bodywt day 0}: * = p < 0.05; ** = p < 0.01

Table 5: Body weight change, males

Sex: Male

 

 

Wgt change

last -x to0(g)

 

[G]

Body wt

change(g)

 

[G]

Body wt

change(g)

 

[G]

-2 - 0

0 - 4

4 - 7

0 ppm

Mean

11.86

6.62

13.06

 

SD

2.82

5.02

2.81

 

N

5

5

5

10 ppm

Mean

10.54

3.48

11.00

 

SD

3.05

3.27

3.24

 

N

5

5

5

100 ppm

Mean

12.12

-0.22

7.96

 

SD

1.60

3.58

2.57

 

N

5

5

5

500 ppm

Mean

8.76

-11.12 **

0.14 **

 

SD

1.66

3.07

3.54

 

N

5

5

5

1000 ppm

Mean

9.66

-32.48 **

-7.54 **

 

SD

2.05

5.39

3.31

 

N

5

5

5

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

Table 6: Body weight change, females

Sex: Female

 

 

Wgt change

last -x to0(g)

 

[G]

Body wt

change(g)

 

[G]

Body wt

change(g)

 

[G]

-2 - 0

0 - 4

4 - 7

0 ppm

Mean

-0.62

4.44

10.74

 

SD

6.62

3.58

6.49

 

N

5

5

5

10 ppm

Mean

4.68

2.52

5.50

 

SD

6.23

5.23

4.62

 

N

5

5

5

100 ppm

Mean

1.62

4.42

4.56

 

SD

8.31

9.42

2.77

 

N

5

5

5

500 ppm

Mean

1.82

1.26

8.08

 

SD

4.97

6.10

1.96

 

N

5

5

5

1000 ppm

Mean

-0.14

-8.10 *

-2.78 **

 

SD

9.85

3.41

2.18

 

N

5

5

5

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

Table 6: Food consumption, males

Sex: Male

Day(s) Relative to Animal Start Date

0 - 4

4 - 7

0 ppm

Mean

21.1

21.8

10 ppm

Mean

19.4

20.3

100 ppm

Mean

19.6

19.3

500 ppm

Mean

14.0

15.5

1000 ppm

Mean

7.5

9.0

Table 7: Food consumption, females

Sex: Female

Day(s) Relative to Animal Start Date

0 - 4

4 - 7

0 ppm

Mean

15.2

14.8

10 ppm

Mean

14.6

14.0

100 ppm

Mean

14.9

13.5

500 ppm

Mean

12.5

14.5

1000 ppm

Mean

6.2

7.8

Table 8: Relative organ weights, males

Sex: Male

 

 

 

 

 

 

 

 

Terminal bodywgt

(g)

 

[G]

Lungs rel.wgt

(g/kg bodywgt)

[G]

Heartrel.wgt

(g/kg bodywgt)

[G]

Adrenalsrel.wgt

(g/kg bodywgt)

[G]

Kidneysrel.wgt

(g/kg bodywgt)

[G]

Liverrel.wgt

(g/kg bodywgt)

[G]

Spleenrel.wgt

(g/kg bodywgt)

[G]

Testesrel.wgt

(g/kg bodywgt)

[G]

0 ppm

Mean

SD N

270.28

16.66

5

4.778

0.223

5

3.161

0.195

5

0.1974

0.0289

5

7.113

0.397

5

39.37

0.68

5

2.123

0.189

5

11.060

1.051

5

10 ppm

Mean SD

N

266.16

16.11

5

4.449

0.261

5

3.074

0.264

5

0.1929

0.0136

5

7.302

0.479

5

38.87

2.23

5

2.216

0.212

5

11.297

0.758

5

100 ppm

Mean SD

N

260.36

16.57

5

4.880

0.313

5

3.158

0.194

5

0.2370

0.0347

5

7.246

0.218

5

39.53

1.94

5

2.173

0.373

5

11.761

0.692

5

500 ppm

Mean

SD N

237.44 *

18.56

5

5.220

0.393

5

3.221

0.166

5

0.2560

0.0516

5

7.299

0.271

5

37.66

1.29

5

2.154

0.345

5

12.878 **

0.915

5

1000 ppm

Mean SD N

210.72 ** 10.57

5

6.235 ** 0.531

5

3.593*0.203 5

0.3132 **

0.0467

5

7.258

0.427

5

36.37*1.55 5

1.760

0.232

5

14.205 ** 0.444

5

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

Table 9: Relative organ weights, females

Sex: Female

 

 

 

 

 

 

 

 

Terminal bodywgt

(g)

 

[G]

Lungs rel.wgt

(g/kg bodywgt)

[G1]

Heartrel.wgt

(g/kg bodywgt)

[G]

Adrenalsrel.wgt

(g/kg bodywgt)

[G2]

Kidneysrel.wgt

(g/kg bodywgt)

[G]

Liverrel.wgt

(g/kg bodywgt)

[G]

Spleenrel.wgt

(g/kg bodywgt)

[G]

Ovaries rel.wgt

(g/kg bodywgt)

[G2]

0 ppm

Mean

SD N

179.60

10.01

5

5.518

0.189

5

3.434

0.116

5

0.3895

0.0695

5

6.974

0.342

5

37.49

0.89

5

2.256

0.243

5

0.4691

0.0414

5

10 ppm

Mean SD

N

178.88

6.97

5

5.230

0.256

5

3.561

0.186

5

0.4016

0.0434

5

7.402

0.517

5

39.61

1.90

5

2.135

0.193

5

0.4768

0.0334

5

100 ppm

Mean SD

N

176.16

7.73

5

5.534

0.231

5

3.420

0.179

5

0.4008

0.0399

5

7.477

0.447

5

39.29

2.02

5

2.093

0.168

5

0.5007

0.1116

5

500 ppm

Mean

SD N

175.18

9.71

5

5.687

0.227

5

3.378

0.100

5

0.4734

0.0867

5

7.148

0.294

5

40.03

3.53

5

2.209

0.109

5

0.4064

0.0352

5

1000 ppm

Mean SD N

151.94 ** 9.01

5

6.897 **

0.524

5

3.933 **

0.199

5

0.4276

0.1323

5

7.737

0.525

5

41.32

1.17

5

1.947

0.199

5

0.3969

0.0547

5

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

[G1] - Ancova/Anova & Dunnett(Log): ** = p < 0.01

[G2] - Kruskal-Wallis & Dunnett on Ranks

Table 10: Macroscopic observations

Removal Reason(s): ALL

 

Number of Animals:

 

0 ppm

 

10

 

ppm

Male 100 ppm

 

500

 

ppm

 

1000

ppm

 

0 ppm

 

10

 

ppm

Female 100 ppm

 

500

 

ppm

 

1000

ppm

5

5

5

5

5

5

5

5

5

5

all organs/tissues

 

 

 

 

 

 

 

 

 

 

no visible lesions

5

5

4

4

-

5

4

5

3

2

lungs

 

 

 

 

 

 

 

 

 

 

incompletely collapsed

-

-

0

0

1

-

0

-

-

0

spots; red

-

-

1

1

4

-

1

-

-

1

lymph node, tracheobronchial

 

 

 

 

 

 

 

 

 

 

enlarged; unilateral

-

-

-

-

1

-

-

-

1

-

thymus

 

 

 

 

 

 

 

 

 

 

small

-

-

-

-

2

-

-

-

-

2

tissue not otherwise specified

 

 

 

 

 

 

 

 

 

 

neck; nodule; white, 2 mm

-

-

-

-

-

-

-

-

1

-

Table 11: Microscopic observations (excerpt)

Removal Reason(s): ALL

 

Number of Animals:

 

0 ppm

 

10

 

ppm

Male 100 ppm

 

500

 

ppm

 

1000

ppm

 

0 ppm

 

10

 

ppm

Female 100 ppm

 

500

 

ppm

 

1000

ppm

5

5

5

5

5

5

5

5

5

5

larynx

 

 

 

 

 

 

 

 

 

 

Examined

4

5

5

5

5

5

5

5

5

5

No Visible Lesions

2

1

4

1

0

4

5

5

1

0

inflammation; granulocytic

0

0

0

3

5**

0

0

0

1

3

.... minimal

0

0

0

1

0

0

0

0

1

1

.... mild

0

0

0

2

5

0

0

0

0

2

metaplasia; squamous

0

0

0

0

4*

0

0

0

0

4*

.... mild

0

0

0

0

4

0

0

0

0

4

ulceration

0

0

0

0

4*

0

0

0

0

0

.... minimal

0

0

0

0

1

0

0

0

0

0

.... mild

0

0

0

0

3

0

0

0

0

0

lungs

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

4

3

3

2

0

2

3

4

4

0

haemorrhage(s); alveolar

0

0

0

0

4*

2

0

0

0

0

bronchus; inflammation; granulocytic

0

0

0

1

4*

0

0

0

1

4*

.... minimal

0

0

0

1

4

0

0

0

1

3

 

nose, level 1

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

5

4

5

3

1

5

3

4

2

3

inflammation; granulocytic

0

1

0

2

4*

0

2

0

3

2

.... minimal

0

1

0

1

2

0

2

0

2

2

.... mild

0

0

0

1

2

0

0

0

1

0

nose, level 2

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

5

5

5

1

0

5

5

5

0

0

inflammation; granulocytic

0

0

0

3

5**

0

0

0

5**

5**

.... minimal

0

0

0

3

0

0

0

0

5

0

.... mild

0

0

0

0

0

0

0

0

0

1

.... moderate

0

0

0

0

2

0

0

0

0

4

.... marked

0

0

0

0

3

0

0

0

0

0

ulceration

0

0

0

0

5**

0

0

0

0

5**

.... mild

0

0

0

0

0

0

0

0

0

2

.... moderate

0

0

0

0

1

0

0

0

0

3

.... marked

0

0

0

0

4

0

0

0

0

0

necrosis

0

0

0

0

5**

0

0

0

0

5**

.... mild

0

0

0

0

0

0

0

0

0

1

.... moderate

0

0

0

0

3

0

0

0

0

1

.... marked

0

0

0

0

2

0

0

0

0

3

metaplasia; squamous

0

0

0

0

3

0

0

0

2

5**

.... minimal

0

0

0

0

0

0

0

0

2

0

.... mild

0

0

0

0

1

0

0

0

0

3

.... moderate

0

0

0

0

2

0

0

0

0

2

nose, level 3

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

5

5

1

1

0

5

5

0

0

0

inflammation; granulocytic

0

0

0

2

5**

0

0

0

1

5**

.... minimal

0

0

0

2

0

0

0

0

1

0

.... mild

0

0

0

0

3

0

0

0

0

4

.... moderate

0

0

0

0

2

0

0

0

0

1

ulceration

0

0

0

0

5**

0

0

0

0

2

.... mild

0

0

0

0

2

0

0

0

0

2

.... moderate

0

0

0

0

3

0

0

0

0

0

necrosis

0

0

0

0

1

0

0

0

0

1

.... mild

0

0

0

0

1

0

0

0

0

1

nose, level 3 (Continued...)

 

 

 

 

 

 

 

 

 

 

olfactory epithelium; atrophy

0

0

4*

3

4*

0

0

5**

5**

4*

.... minimal

0

0

4

1

0

0

0

5

5

2

.... mild

0

0

0

2

2

0

0

0

0

2

.... moderate

0

0

0

0

2

0

0

0

0

0

metaplasia; squamous

0

0

0

1

4*

0

0

0

0

5**

.... mild

0

0

0

1

2

0

0

0

0

1

.... moderate

0

0

0

0

2

0

0

0

0

4

nose, level 4

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

4

5

2

0

0

5

5

0

0

0

inflammation; granulocytic

0

0

0

2

5**

0

0

0

2

5**

.... minimal

0

0

0

2

0

0

0

0

2

0

.... mild

0

0

0

0

4

0

0

0

0

5

.... moderate

0

0

0

0

1

0

0

0

0

0

ulceration

0

0

0

0

5**

0

0

0

0

4*

.... minimal

0

0

0

0

0

0

0

0

0

1

.... mild

0

0

0

0

3

0

0

0

0

3

nose, level 4 (Continued...)

 

 

 

 

 

 

 

 

 

 

.... moderate

0

0

0

0

1

0

0

0

0

0

.... marked

0

0

0

0

1

0

0

0

0

0

olfactory epithelium; atrophy

0

0

3

4*

4*

0

0

4*

5**

3

.... minimal

0

0

2

2

3

0

0

3

1

2

.... mild

0

0

1

2

1

0

0

1

4

1

metaplasia; squamous

0

0

0

0

5**

0

0

0

0

5**

.... minimal

0

0

0

0

1

0

0

0

0

0

.... mild

0

0

0

0

4

0

0

0

0

4

.... moderate

0

0

0

0

0

0

0

0

0

1

nose, level 5

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

5

5

4

3

0

5

5

5

3

0

inflammation; granulocytic

0

0

0

0

5**

0

0

0

0

4*

.... minimal

0

0

0

0

1

0

0

0

0

2

.... mild

0

0

0

0

4

0

0

0

0

2

ulceration

0

0

0

0

2

0

0

0

0

2

.... minimal

0

0

0

0

1

0

0

0

0

0

.... mild

0

0

0

0

0

0

0

0

0

2

.... moderate

0

0

0

0

1

0

0

0

0

0

nose, level 5 (Continued...)

 

 

 

 

 

 

 

 

 

 

metaplasia; squamous

0

0

0

0

5**

0

0

0

0

5**

.... minimal

0

0

0

0

1

0

0

0

0

0

.... mild

0

0

0

0

4

0

0

0

0

5

nose, level 6

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

5

5

5

4

0

5

5

5

5

3

trachea/bronchi

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

4

4

5

1

0

5

5

5

1

1

inflammation; granulocytic

0

0

0

3

5**

0

0

0

0

4*

.... minimal

0

0

0

1

0

0

0

0

0

1

.... mild

0

0

0

2

5

0

0

0

0

3

inflammation; mixed

0

0

0

1

0

0

0

0

2

0

.... minimal

0

0

0

1

0

0

0

0

1

0

Fisher's Exact: * = p < 0.05; ** = p < 0.01

Conclusions:
Under the conditions of this 7-day concentration range-finding study, inhalation exposure to the test substance at target concentrations of 10, 100, 500 and 1000 ppm resulted in the following treatment-related changes:
• At 1000 ppm, marked toxicity was observed; in nearly all animals, dyspnea, sniffing, irregular respiration, hunched posture, ataxia, blepharospasm, piloerection and hypoactivity were observed. Body weight of males exposed to the top concentration were 22% lower than controls (on day 7); for females the reduction in body weight was 15%. Relative weight of the heart and lungs was increased in male and female animals. When assessing histopathology, severe olfactory epithelial atrophy was observed in the nasal tissues. Milder effects were observed in the larynx, trachea/bronchi and lungs, mainly of inflammatory nature.
• At 500 ppm, transient clinical abnormalities (dyspnea, shallow breathing, hunched posture and piloerection) were observed in males especially. Minimal to mild atrophy of the nasal olfactory epithelium was observed in most animals when assessing nose tissue levels 3 and 4 (out of the 6 levels assessed).
• At 100 ppm, minimal atrophy of the nasal olfactory epithelium was still observed in most animals, with 2 cases of mild atrophy observed at a single level of the nose in 2 animals.
• At 10 ppm, no exposure-related toxicity was seen. Thus, the No-Observed-Adverse-Effect- Concentration (NOAEC) in this range finding study was placed at 10 ppm (target concentration).

Based on these observation, the target concentration for the main study were selected at 10, 50, and 250 ppm. Sub-acute exposure to the test substance at 250 ppm is anticipated to result in clear respiratory toxicity (without causing lethality or severe distress), while at 10 ppm exposure-related adverse changes are no longer expected.
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Name1 : Dimethylethylamine
Chemical name 1 : Ethyldimethylamine; N,N-Dimethylethylamine
CAS registry number1 : 598-56-1
Molecular formula1 : C4H11N
Structural formula1 :
Molecular weight1 : 73.13 g/mol
Batch/lot number1 : SAMP160321
Purity1 : 99.41%
Solubility in water1 : completely soluble; exothermic reaction!
Melting point1 : -140 °C
Boiling point1 : 36.3 °C
Vapor pressure 1 : 527 hPa at 20° C
Density 1 : 660 kg/m3 at 20 °C
Viscosity1 : 0.67 mPa.s (OECD Test Guideline 114)
Flash Point1 : -25°C closed cup (ISO 3679)
Quantity received : 6.75 kg
Supplier : Arkema
Approved for use until1 : 20 November 2018
Storage conditions : ambient temperature (15-25°C)
Date of receipt : 23 November 2017
1Characteristics provided by the sponsor
The Triskelion dispense reference number is 17025D.
Species:
rat
Strain:
Wistar
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 regulatory authorities. Young adult, male and female Wistar outbred (Crl:WI(Han)) rats were obtained from a colony maintained under specific pathogen free (SPF) conditions by Charles River Laboratories. The Wistar rat strain was used because it is routinely used at the test facility for this type of study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
The age of the rats was about 7-8 weeks on the day of randomization (8 January and 29 March 2018 for animals of the range finding and main study, respectively). Body weight at allocation was within ±20% of the mean weight for each sex. Mean body weights at the start of exposure (day 0) in the range finding study were 251 and 166 grams for male and female animals, respectively. Mean body weights at the start of exposure in the main study were 235 and 170 grams for male and female animals, respectively.

Upon arrival on 21 March 2018, the rats (33 males and 33 females) were taken in their unopened shipping containers to a quarantine room (animal room 5.1.14) and were checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from five randomly selected animals. On 23 March 2018, the results of the serological examinations were received and indicated an acceptable microbiological status. The animals were subsequently released for experimental use, and were transferred to their definitive room (animal room 6.0.06). The duration of the acclimatization period to the laboratory conditions prior exposure (period between arrival and the start of the exposure period) was 13 days.

Shortly before initiation of exposure (on 29 March 2018), the animals were allocated to the various groups by computer randomization proportionally to body weight (males and females separately). The surplus animals (3 males, 3 females) were kept in reserve to serve as sentinels. These animals were not used in the present study.

Animal husbandry
Animal room
From their arrival, the animals were housed under conventional conditions in one animal room separated by sex. No other test system was housed in the same room during the study. The room was ventilated with about 10 air changes per hour and was maintained at a temperature of 20-24oC and a relative humidity of 45-65%. The upper limit of relative humidity was, however, higher than 65% for short periods of time because of wet cleaning activities. Relative humidity was below 45% on 31 March and 3 April 2018 (briefly, with a minimum of 44%) and on multiple occasions for prolonged durations in the period 5 April – 24 May 2018 (minimum of 39%). Lighting was artificial with a sequence of 12 hours light and 12 hours dark.

Caging
During exposure, the animals were housed individually in the inhalation unit. Immediately after each exposure, the animals were returned to their home cages. When not exposed, the animals were housed five animals to a cage, separated by sex. All animals were housed in macrolon cages with wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany) as bedding material and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. The cages and bedding were changed at least weekly.

Food and drinking water
Food was provided ad libitum from the arrival of the animals until the end of the study, except during exposure and – for animals of the main study – the overnight fasting period before sacrifice. The animals received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Whitham, England). Each batch of VRF1 (FG)) diet is analyzed by the supplier for nutrients and contaminants. The food was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the feeders was replaced with fresh portions once weekly and filled up as needed.

Drinking water was provided ad libitum from the arrival of the animals until the end of the study, except during exposure. Each cage was supplied with 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. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the test facility. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of the
test facility for a limited number of variables.

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Exposure equipment
The animals were exposed to the test atmosphere in nose-only exposure units, in an illuminated laboratory room different from the room where the animals were housed. Animals of groups 2, 3, 4 and – for the range finding – 5 were exposed in inhalation chambers consisting of a cylindrical aluminium column, surrounded by a transparent cylinder (a modification of the chamber made by ADG Developments Ltd., Codicote, Hitchin, Her ts, SG4 8UB, United
Kingdom). The column had a volume of 46.7 L and consisted of a top assembly with the entrance of the unit, a mixing section, two rodent tube sections and at the bottom the base assembly with the exhaust port. Each rodent tube section had 20 ports for animal exposure. Control animals (group 1) were exposed to clean air in a polypropylene nose-only inhalation chamber with a volume of 48.2 L (manufactured by P. Groenendijk Kunststoffen BV) which was very similar in construction to the aluminium chambers described above.

Empty ports were used for test atmosphere sampling (for analysis of the actual concentration) and measurement of oxygen, carbon dioxide, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Animals were rotated with respect to their position in the column every week (main study) or two days (range finding study). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. Habituation to the restraint in the animal holders was not performed because in our experience habituation does not help to reduce possible stress (Staal et al., 2012).

In our experience, the animal’s body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder, which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animals’ thorax to the nose was avoided. The unit was illuminated externally by normal laboratory fluorescent tube lighting. The total air flow through the unit was at least 1 liter/min for each rat. The air temperature and relative humidity in the unit were maintained at 22 ± 3°C and between 30 and 70%, as far as possible.

Generation of the test atmosphere
The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. A schematic diagram of the generation and the exposure system is presented in Figure 1. To generate the test atmospheres, a continuous flow of liquid test material3, controlled by a peristaltic pump (Minipulse 3, Gilson, Velliers le Bel, France) was allowed to evaporate in a mass flow controlled (Bronkhorst Hi Tec, Ruurlo, the Netherlands) stream of compressed dry air, by directing it through a glass evaporator which was kept at a constant temperature of 48°C by circulating heated water. The resulting single stream of concentrated vapor was led through a condense trap (not used during the range finding study) and was subsequently divided for the different groups using mass flow controllers (Bronkhorst Hi Tec) and mixed with a controlled stream of humidified compressed air via an eductor (Fox Valve Development Corp., Dover, NJ, USA). The eductors were calibrated by measuring the total air flow at a range of driving air pressures of the eductors encompassing the driving pressures used during the study; the driving air pressure was used to monitor the total flow. The resulting test atmosphere was led to the inlet at the top of the exposure chamber and directed downward towards the noses of the animals; the atmosphere was exhausted at the bottom. As of 9 April 2018, a PI feedback system was installed to automatically control the feedrate of the peristaltic pump, based on the measured actual concentration (see paragraph 4.10.1). The feedback system took into account the proportional (P) and the integrated deviations (I) of the concentrations from the setpoint.

The exposure chamber for the control animals (group 1) was supplied with a stream of humidified compressed air only, which was controlled by a reducing valve and measured by 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of exposure conditions
Actual concentration
The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (groups 2 and 3 of the main study and group 3 of the range finding study: Ratfisch RS55T, Munich, Germany; remaining groups: 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 Data acquisition system (range finding study: CAN transmitters, G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany; main study: MyriaNed, van Mierlo Ingenieursbureau BV, Eindhoven, the Netherlands). 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.

Prior to the first exposure, the output of the flame ionization detector of the TCA was calibrated using gas sample bags. To this end, sample bags were filled with an accurate (mass flow controlled) volume of air and an accurate (weighed) amount of test material, injected into the bag through a septum. Three concentrations were thus prepared (at least in duplicate) – at approximately 80%, 100% and 120% of the target concentration of each group – and analyzed by the TCA. Linear relations were found between the response of the analyzers and the concentration of the test material.

The calibrations were checked weekly during the study. To this end, gas sample bags were prepared at each target concentration as described above, and were subsequently analyzed by the TCA. If the measured concentration deviated more than 5% from the calculated concentration, the calibration check was repeated. If the deviation was more than 5% at the re-check, a complete re-calibration was to be carried out (which was not necessary during the range finding nor the main study).
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
5 days per week, over a 28-day study period (20 exposure days in total)
Dose / conc.:
0 ppm (analytical)
Dose / conc.:
10 ppm (analytical)
Remarks:
30 mg/m3
Dose / conc.:
49.7 ppm (analytical)
Remarks:
149.1 mg/m3
Dose / conc.:
249 ppm (analytical)
Remarks:
747 mg/m3
No. of animals per sex per dose:
The 28-day study comprised four test groups of five male and five female rats each, i.e. a control group exposed to clean air and three groups exposed to different concentrations of the test material.

Five additional males and 5 additional females were included in the control and high-concentration group, which were exposed similarly and kept for a recovery period of 28 days after the last exposure.
Control animals:
yes, concurrent no treatment
Details on study design:
7-day concentration range finding study:
The range finding study consisted of five test groups of five male and five female rats each, i.e. a control group exposed to clean air, and four groups exposed to different concentrations of the test material. The highest concentration was intended to result in clear toxicity but not death or moribundity. The lowest concentration was intended to produce little or no evidence of toxicity. The animals of the control group were handled identically as those of the other groups, except for exposure to the test material. The animals were exposed for 6 hours per day, 5 days per week, over a 7-day study period (5 exposure days in total) and sacrificed on the day after the last exposure. The date of the first exposure (10 January 2018) was named day 0.

Concentrations (ppm): 0, 10, 100, 500 and 1000

Selection of target concentrations for the concentration range finding study were based on the 4h LC50 that was found to be ca. 3400 ppm; no mortality was observed at a lower concentration of 2300 ppm. Although no repeated-dose inhalation toxicity data were available for Dimethylethylamine, there were data for the toxicity of the comparable substance Trimethylamine (CAS no. 75-50-3). The 4h LC50 was comparable at ca. 2400-3500 ppm. In a subacute study, mild toxicity was observed at the low concentration of 75 ppm, while mild toxicity was observed at a concentration as low as 10 ppm in a chronic study (all information available at http://www.echa.eu); thus, to enable identification of a NOAEC and characterize the concentration-response relationship, a broad concentration range was chosen for this range finding study.


Animal allocation
7-day range finding study
27 male and 27 female rats were ordered, and upon arrival on 20 December 2017 they were taken in their unopened shipping containers to a quarantine room (animal room 5.1.20) and were checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from four randomly selected animals. On 22 December 2017, the results of the serological examinations were received and indicated an acceptable microbiological status. The animals were subsequently released for experimental use, and they were transferred to their definitive room (animal room 6.0.06) on the same day. The duration of the acclimatization period to the laboratory conditions prior exposure (period between arrival and the start of the exposure period) was 21 days.

On 8 January 2018, shortly before initiation of exposure of the rats, they were allocated to the various groups by computer randomization proportionally to body weight. . The surplus animals (2 males, 2 females) were kept in reserve to serve as sentinels. These animals were not used in the present study.

28-day main study
Upon arrival on 21 March 2018, the rats (33 males and 33 females) were taken in their unopened shipping containers to a quarantine room (animal room 5.1.14) and were checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from five randomly selected animals. On 23 March 2018, the results of the serological examinations were received and indicated an acceptable microbiological status. The animals were subsequently released for experimental use, and were transferred to their definitive room (animal room 6.0.06). The duration of the acclimatization period to the laboratory conditions prior exposure (period between arrival and the start of the exposure period) was 13 days.

Shortly before initiation of exposure (on 29 March 2018), the animals were allocated to the various groups by computer randomization proportionally to body weight (males and females separately). The surplus animals (3 males, 3 females) were kept in reserve to serve as sentinels. These animals were not used in the present study.

4.6 Identification
The study was identified as Triskelion study number 21124, with a subcode /01 for the range finding study and /02 for the main study.

Before and during allocation, the individual rats were identified by a transient mark on their tail.

After allocation, they were identified by an identification number (even for males and odd for females), using subcutaneous transponders. During the study each group of rats was coded by a number and a color. Each cage was provided with a card showing the color code, the animal identification numbers, the group number and the study number.
Positive control:
None
Observations and examinations performed and frequency:
Clinical observations
Animals were observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. The observations included – but were not limited to – the signs listed in Annex 6. 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 hindered 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 examination (28-day sub-acute study)
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 groups (on day 23). Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate. Since no evident exposure-related ocular changes were observed, eye examinations were not extended to the animals of the intermediate concentration groups at the end of the exposure period, or to animals of the recovery groups.

Body weights
In the range finding study, the body weight of each animal was recorded 2 days before the start of the exposure. These pre-test weights served as a basis for animal allocation. Subsequently, the animals were weighed prior to the first exposure on day 0, on day 4 of exposure and on their scheduled sacrifice data in order to calculate the correct organ to body weight ratios.

In the main study, the body weight of each animal was recorded 5 days before the start of exposure. These pre-test weights served as a basis for animal allocation. Subsequently, the animals of the main study were weighed prior to exposure on the first day (day 0), and twice a week thereafter. 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.

Food consumption
Food consumption of the animals was measured per cage by weighing the feeders. The results are expressed in g per animal per day. Food consumption was measured twice weekly for animals of the range finding study. For animals of the main study, food consumption was measured once weekly.

Estrus cycle evaluation (28-day sub-acute study)
Vaginal smears to evaluate the estrus cycle length and normality were made daily in the three weeks prior to sacrifice, including the day of sacrifice, in all female animals of the main study. Since no exposure-related abnormalities were observed in animals of the main study, estrus cycle evaluation was not extended to animals of the recovery groups.
Sacrifice and pathology:
Sperm analysis (28-day sub-acute study)
Epididymal sperm motility, count and morphology At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all male animals from both the main and recovery groups (30 animals in total). For this purpose the cauda epididymis was dissected, weighed and thereafter minced in M199 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) were measured for all males, using the Hamilton Thorne Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained, and two hundred spermatozoa of this smear were examined for morphology.

Testicular sperm count
At scheduled necropsy, the left testis of all males were placed on dry ice and subsequently stored in a freezer (<-70°C) for later determination of the number of homogenization-resistant spermatids. The testes to be analysed were thawn just before further processing. Following removal of the tunica albuginea, the testicular parenchyma were weighed, minced and homogenized in Saline Triton X-100 solution. Following DNA-staining, the homogenizationresistant sperm heads were enumerated using the IVOS. The daily sperm production was calculated. The evaluation of homogenization-resistant spermatids was performed in the males of the control and high-concentration groups from both the main and recovery study. Since no exposure-related changes were observed in animals of the high-concentration group, the evaluation of homogenization-resistant spermatids was not extended to animals of the intermediate-concentration groups.

Hematology (28-day sub-acute study)
Hematology was conducted at the end of the treatment period on all surviving animals of the main groups. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anesthesia at sacrifice. Citrate (for prothrombin time) or EDTA (for other parameters) were used as anticoagulant. Blood samples were discarded after analysis. In each sample the following determinations were carried out.
- hemoglobin
- packed cell volume
- red blood cell count
- reticulocytes
- total white blood cell count
- differential white blood cell count
(lymphocytes, neutrophils, eosinophils, basophils and monocytes)
- prothrombin time
- thrombocyte count (platelet count)

The following parameters were calculated:
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin (MCH)
- mean corpuscular hemoglobin concentration (MCHC)

Since (possible) exposure-related changes were observed in animals of the main groups, investigation of all hematology parameters (except prothrombin time) was extended to animals of the recovery groups.

4.11.8 Clinical chemistry (28-day sub-acute study)
Clinical chemistry was conducted at the end of the exposure period on all surviving rats after overnight fasting, at the same time blood samples for hematology were collected. Blood samples were taken from the abdominal aorta of the rats whilst under pentobarbital anaesthesia. The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. Plasma samples were stored frozen (<-18°C) until analysis and
discarded afterwards.
- alkaline phosphatase activity (ALP) - bilirubin (total)
- aspartate aminotransferase activity (ASAT) - cholesterol
- alanine aminotransferase activity (ALAT) - triglycerides
- gamma glutamyl transferase activity (GGT) - calcium (Ca)
- total protein - sodium (Na)
- albumin - potassium (K)
- ratio albumin to globulin (calculated) - chloride (Cl)
- urea - inorganic phosphate (PO4)
- creatinine - thyroxine (T4)
- glucose (fasting)

Since (possible) exposure-related changes were observed in animals of the main groups, investigation of clinical chemistry parameters was extended to animals of the recovery groups.

Pathology
Sacrifice, organ weights and macroscopic examination
At the end of the exposure period, the animals were sacrificed 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 28-day post-exposure recovery period included in the sub-acute study. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes.

7-day concentration range finding study
The following organs of all 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 weights and the terminal body weight:
- heart
- adrenals
- kidneys
- liver
- spleen
- testes
- ovaries
- lungs with trachea

28-day sub-acute study
The following organs of all 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 weights and the terminal body weight:
- adrenals
- brain
- heart
- kidneys
- liver
- lungs (left lung only, because of lavage of the other lobes)
- spleen
- testes
- ovaries
- thymus

For assessment of fertility in male rats of the main groups (20 animals in total), the following additional weights were determined:
- epididymides
- prostate
- seminal vesicles

Since (possible) exposure-related changes were observed in animals of the main groups, the seminal vesicles (+ coagulation glands), testes, epididymides (all three organs for males only) and thymus (both sexes) were also weighed in animals from the recovery groups of the main study; the other organs included for main study animals were not weighed.

Bronchoalveolar lavage and measurements (28-day sub-acute study)
The lungs of all animals of the main groups were lavaged at necropsy according to a standardized method. In short: the right half of the lungs (after binding off the left lung lobe, used for histopathology) from these animals was rinsed three times with a single volume of 26.7 ml saline per kg body weight (one value for each group based on mean body weight). The final amount of lung lining fluid and cells collected was weighed and retained on ice. The
bronchoalveolar lavage cells were recovered by centrifugation (250xG) for 5 minutes. The temperature control of the centrifuge was set at 4ºC. Each cell pellet thus obtained per animal was resuspended in 0.5 ml saline and used for total white blood cell numbers, viability and cell differentials. The supernatant was stored frozen (<-18°C) until analysis of biochemical parameters. BAL fluid samples were discarded after analysis.

Cellular determinations
Total white blood cell numbers were counted using an AdVia 2120i analyzer (Siemens N.V., the Netherlands; Reference: “Training manual 04/11/99, chapter 5 Impedance”.) The number of viable cells was determined using an acridine orange / ethidium bromide staining method in combination with fluorescent microscopic evaluation. The cytospins for cell differentials were made using a Cyto-Tek (Sakura, Netherlands) and stained by May-Grunwald Giemsa. The differential cells (monocytes, macrophages, neutrophils, eosinophils, lymphocytes) were evaluated by light microscopy (absolute numbers were calculated from total white blood cell number and percentage distribution of the different cell types).

Since exposure-related changes were observed in animals of the main groups, investigation of bronchoalveolar lavage parameters was extended to animals of the recovery groups of the sub-acute study.


Histopathology
7-day concentration range finding study
For histopathological examination, samples of the following tissues and organs from all animals, and all gross lesions were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of Formalin). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to ensure fixation). Tissues were processed and examined using the same methods as described below for the relevant tissues examined during the 28-day study. The carcass containing any remaining tissues was retained in the fixative until completion of the histopathological examination and then discarded.
- kidneys
- nasal turbinates
- larynx
- trachea
- lungs
- tracheobronchial lymph nodes

All preserved tissues of all animals of the control and top-concentration groups were examined histopathologically (by light microscopy). Since exposure-related changes were observed in the nasal tissues (all 6 levels), larynx, trachea and lungs of the top-concentration group, histopathological examination of these tissues was extended to animals of the intermediate concentration groups of the range finding study.

28-day sub-acute study
For histopathological examination, samples of the following tissues and organs from all animals, and all gross lesions were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of Formalin). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to insure fixation).
- adrenals
- bone marrow
- brain (including sections of cerebrum, cerebellum, and medulla/pons)
- eyes (with specific focus on adverse corneal changes)
- heart
- kidneys
- liver
- esophagus
- ovaries
- seminal vesicles
- spinal cord (cervical, mid-thoracic, and lumbar)
- spleen
- stomach
- testes (including testicular staging)
- thymus
- thyroid
- tracheobronchial lymph nodes
- uterus
- respiratory tract including larynx, trachea, left lung lobe (the right lobes were lavaged) and
nasopharyngeal tissues
- all gross lesions

The carcass containing any remaining tissues was also retained in Formalin, but discarded after completion of the histopathological examination.

Slide preparation
Tissues to be examined were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. Unless required for histopathological examination, the tissues of the animals of the intermediate concentration groups (groups 2 and 3) were not processed, except for the noses of animals of groups 2 and 3 which were decalcified and embedded in paraffin concurrently with the noses of the animals of groups 1 (control) and 4 (high concentration).

Histopathological examination
All preserved tissues of all animals of the control and high-concentration main groups were examined histopathologically (by light microscopy). In addition, all gross lesions observed in rats of the intermediate-concentration main groups were examined microscopically. Histopathology was subjected to a peer review system.

The nasopharyngeal tissues were examined at six levels (Woutersen et al., 1994; an illustration of these levels is shown in Annex 9) with one level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT), the larynx at three levels (one level to include the base of the epiglottis). The trachea was examined at three levels (including the bifurcation, and one longitudinal section through the carina), and the left lung lobe at three levels. When examining the eye, particular focus was directed towards assessment of corneal changes, which have been observed in exposed workers who have experienced “blue haze”, associated with
corneal swelling.

Since exposure-related changes were observed in the nasal tissues of animals of the highconcentration group, histopathological examination of these tissues was extended to animals of the intermediate concentration main groups (nose levels 2-6) and to animals of the recovery groups (nose levels 1-6) of the 28-day sub-acute study.
Statistics:
Ancova/Anova & Dunnett
Pretreatment body weight, body weight after initiation of treatment, clinical pathology (hematology, clinical chemistry), bronchoalveolar lavage parameters, organ weights, sperm motility
(numerical), sperm count, testicular sperm count.
Food consumption - no statistics were applied (only one cage per sex per group).
Kruskal-Wallis & Wilcoxon: Estrus cyclicity: mean length of the longest cycle, number of complete cycles in the test period; sperm motility (expressed as %), sperm morphology
Chie-squared & Fisher's Exact: Incidences of histopathological changes; Estrus cyclicity: number of acyclic females, number of animals with prolonged estrus period
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No exposure-related clinical abnormalities were observed. The few signs noted were considered unrelated to the exposure to the test material. Abnormalities of the skin or fur (sparsely haired areas, encrustations) were observed in a few animals across the groups. These are common findings, possibly caused by slight movement of the animals in the restraining tubes during exposure, resulting in slight irritation of the skin.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male animals of the high-concentration group showed a statistically significantly lower body weight than controls from day 3 until the end of the exposure period (up to ~10% lower average body weight by day 24), which was the result from body weight loss or decreased growth on days of exposure; normal growth was observed during periods including the exposure-free weekends. The difference in body weight compared to control animals gradually decreased during the recovery period. A trend towards decreased body weight gain was also observed in females of the high-concentration group and in males of the mid-concentration group during the first ~2 weeks of exposure, but statistical significance was not reached.

PLEASE REFER TO ATTACHED TABLES - 'Body weight' and Body weight change'
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Although statistical analysis could not be performed (as only one cage per sex per group was used), food consumption of male animals of the high-concentration group was slightly lower (maximally approximately 15%) than controls throughout the exposure period; normal food intake was observed during the recovery period. Food consumption in females of the high-concentration group also seemed slightly decreased (maximally approximately 10%) during the first half of the exposure period, when compared to controls. No treatment-related changes in food intake were observed in animals of the low- and midconcentration group.

PLEASE REFER TO ATTACHED TABLE - 'Food consumption'
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Ophthalmoscopic examination near the end of the exposure period revealed unilateral focal corneal opacity in two (of five) male animals of the high-concentration main group. This – usually transient, reversible – lesion is occasionally observed as a background finding, possibly related to the nose-only mode of exposure in restraining tubes (sometimes causing minor superficial damage to the cornea, because the animal’s head may be somewhat compressed against the front of the tube). For this reason, and because the lesion was observed only focally (rather than diffusely throughout the cornea), in just two animals of the high-concentration group, and was not associated with any microscopic changes in the eye, a relation with the exposure to the test material was considered to be highly unlikely. No further ophthalmoscopic abnormalities were observed at the end of the exposure period.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Analysis of hematology parameters revealed the following statistically significant differences between animals of the main groups exposed to the test material and unexposed controls:
- Decreased absolute number of eosinophils in males of the high-concentration group. This finding was not accompanied by any changes in other white blood cell parameters, or by a shift in the percentage distribution of differential white blood cell numbers and might be a consequence of stress.
- Increased packed cell volume (PCV) in males of the low-concentration group, which was considered to be a chance finding (unrelated to the exposure), because a concentration-response relationship was absent.
There were no statistically significant changes in coagulation parameters of animals of the main groups.

Investigation of hematology parameters in animals of the recovery groups, performed in response to the statistically significant differences observed in animals of the main groups, did not reveal any exposure-related changes. In the absence of any corresponding changes at the end of the exposure period or any corroborative findings in animals of the recovery groups, a slightly increased mean corpuscular volume (MCV) in males of the high-concentration (without any changes in parameters from which MCV is calculated, i.e. PCV and total red blood cell numbers) and slightly lower lymphocyte and total white blood cell numbers in females of the
high-concentration recovery group were considered to be chance findings, unrelated to the exposure to the test material.

PLEASE REFER TO ATTACHED TABLES - 'Red blood cell and coagulation parameters (main and recovery)', 'Total and differential white blood cell counts (main and recovery)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Analysis of clinical chemistry parameters revealed the following statistically significant differences between animals of the main groups exposed to the test material and unexposed controls:
- Decreased plasma activity of alkaline phosphatase (ALP) in males of all dose groups. This difference was considered to be a chance finding (unrelated to the exposure), caused by a few relatively high values recorded for concurrent control animals, which was substantiated by the results obtained in recovery animals (showing a control range of plasma ALP activity similar to exposed animals of the main groups). Moreover, an increase – rather than a decrease – in ALP activity is commonly associated with pathological changes. Thus, the apparent decrease in ALP activity in exposed males of the main groups was considered to be of no toxicological relevance.

- Increased thyroxine (T4) concentration in plasma of females of the high-concentration group. Though increased relative to concurrent controls, the average T4 concentration (466.22 ± 133.43 ng/mL) was within the range of recent historical controls and in line with results obtained in animals of the recovery group.

- Increased plasma sodium (Na) concentration in females of the mid-concentration group, which was considered to be a chance finding, because a concentration-response relationship was absent.

Since statistically significant differences were observed in animals of the main groups, clinical chemistry parameters were also examined in animals of the recovery groups. No exposure-related changes were observed at the end of the recovery period. A few statistically significant differences in females of the high-concentration group when compared to concurrent controls (slightly lower ALP activity, slightly lower average plasma albumin concentration and – consequently – a decreased albumin/globulin ratio) were considered chance findings, since corresponding changes were not observed at the end of the exposure phase.

PLEASE REFER TO ATTACHED TABLES - 'Clinical chemistry (main and recovery)'
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):
Organ weight data obtained at necropsy of animals of the main groups at the end of the exposure period showed a statistically significant increase in relative weight of the testes and adrenals in males of the high-concentration group. Since the absolute weight was unchanged, this change is considered secondary to the reduced body weight. Stress might have enhanced the effect on adrenal weights. There was a decrease in absolute and relative weight of the thymus in females of the high-concentration group. Thymus weight also seemed decreased in males of the high-concentration group, but statistical significance was not reached. No exposure-related changes were observed in the weight of selected organs (seminal vesicles, testes, epididymides, and thymus) of animals sacrificed at the end of the recovery period.
In the absence of any changes after the recovery phase and no histopathological findings, a lower thymus weight was likely stress related caused by inhalation of a caustic substance, and was considered treatment-related but non-adverse.

A few statistically significant changes in organ weight of females of the low-concentration group (decreased absolute spleen weight, decreased absolute and relative thymus weight) were considered chance findings, because a concentration-response relationship was absent.

PLEASE REFER TO ATTACHED TABLES - 'Absolute organ weights (main and recovery)' 'Relative organ weights (main and recovery)'
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy, no exposure-related macroscopic changes were observed in the animals of the main and recovery groups. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in the different groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic evaluation revealed an exposure-related, concentration-dependent increase of degeneration of the respiratory and olfactory epithelium in the nasal tissues of animals of the mid- and high-concentration groups sacrificed at the end of the exposure period. In the highconcentration group, all animals showed minimal to mild degeneration of the respiratory epithelium in the rostral parts of the nose (level 2) and mild to moderate degeneration of the olfactory epithelium in the dorsocaudal parts of the nose (levels 3-6). The incidence and severity of these lesions was lower in animals of the mid-concentration group, with minimal to mild degeneration of the respiratory epithelium observed at level 2 in 4/10 animals, and minimal to mild degeneration of the olfactory epithelium observed at levels 3 (10/10 animals), 4 (9/10 animals) and 5 (2/10 animals). No exposure-related nasal pathology was observed in animals of the low-concentration group.

The other organs and tissues did not reveal any exposure-related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to the exposure.

Because of the histopathological changes observed in animals of the main groups at the end of the exposure period, microscopic examination of the nasal tissues was extended to animals of the recovery groups (control and high-concentration). Substantial – but not complete – recovery of the nasal lesions was observed at the end of the 4-week exposure-free recovery period. Minimal to mild degeneration of the olfactory epithelium was still observed in 7/10 animals at level 3 and in 1/10 animals at level 4 of the nasal tissues of animals of the high-concentration group. No exposure-related changes were observed in the respiratory epithelium – or in other
parts – of the nasal tissues of animals of the recovery groups.

PLEASE REFER TO THE ATTACHED TABLES - 'Microscopic observations (main and recovery)'
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Bronchoalveolar lavage and measurements
Analysis of BAL parameters at the end of the exposure period revealed an increase in the absolute number of macrophages in BAL fluid of exposed males (and consequently a trend towards increased total / viable cell numbers), which reached the level of statistical significance in males of the high-concentration group. These findings were not associated with any changes in the percentage distribution of white blood cells (BAL fluid normally contains ~100% macrophages in healthy animals). No exposure-related changes in cell differentials were observed in female animals. Investigation of biochemical parameters revealed a slight, but statistically significant increase in gamma-glutamyltransferase (GGT) activity in BAL fluid of females of the mid- and high-concentration group; no changes were found in male animals at the end of the exposure period.

Analysis of BAL parameters at the end of the recovery period did not show any statistically significant differences between exposed animals an unexposed controls, although a trend towards an increased absolute number of macrophages and total cells was still observed in males of the high-concentration group.

PLEASE REFER TO ATTACHED TABLES - 'Bronchiolalveolar lavage: biochemical determinations (main and recovery)' 'Brochiolar lavage: volume and absolute cell count (main and recovery)' 'Bronchiolar lavage: relative cell count (main and recovery)'
Key result
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
49.7 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
10 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no

Estrus cyclicity

Estrus cycle evaluation during the last three weeks of the exposure period did not reveal any exposure-related changes in cycle length or normality of the cycle.

Sperm analysis

Epididymal sperm count

No exposure-related statistically significant differences were observed on epididymal sperm count between the control- and exposure groups.

Epididymal sperm morphology

No exposure-related changes were observed in epididymal sperm morphology. A statistically significantly increased occurrence of ‘small hooks’ in males of the high-concentration recovery group was considered to be a chance finding, because corresponding changes at the end of the exposure period – or any corroborative changes in sperm morphology – were absent.

Epididymal sperm motility

No exposure-related statistically significant differences were observed on epididymal sperm motility. The statistically significantly increased average curvilinear velocity (VCL) in males of the high-concentration recovery group (relative to concurrent controls, but in the same range as controls of the main groups) was considered to be a chance finding, because a similar change was not observed in animals sacrificed at the end of the exposure period (also, a decrease – rather than an increase – in VCL in commonly associated with adverse sperm changes).

Testicular sperm count

There were no statistically significant differences in testicular sperm count parameters between the control- and the high-concentration group.

Analysis of the exposure conditions

Actual concentration

The overall average actual concentrations (± standard deviation) of Dimethylethylamine in the test atmospheres, as determined by total carbon analysis, were 10.0 (± 0.2), 49.7 (± 0.8) and 249.0 (± 4.0) ppm for the low-, mid- and high-concentration groups, respectively. These concentrations were very close to the respective target concentrations of 10, 50 and 250ppm.

Time to attain chamber equilibration (T95)

The time to reach 95% of the steady state concentration (T95), based on chamber volume and the average total flow, was calculated to be 5.1 – 5.7 minutes.

Generation efficiency

The overall average (± standard deviation) generation efficiency, as calculated from the actual concentration, flows and test material consumption, was 84.0 (± 5.3)%. The efficiency of a vapor test atmosphere generation is commonly close to 100%. The slightly lower (than 100%) efficiency of the current test atmosphere generation was probably related to minor losses of test material due to condensation in the ‘liquid catch’, which was installed after the evaporator to prevent condensation down the line.

Total flow, temperature, relative humidity, oxygen and carbon dioxide concentration

The average (± standard deviation) total flows of test atmosphere were 25.9 (± 0.4), 24.5 (± 0.0), 25.2 (± 0.0) and 27.4 (± 0.2) L/min for the control, low-, mid- and high-concentration groups, respectively.

The average temperature (± standard deviation) was 23.05 (± 0.26), 22.99 (± 0.25), 22.81 (± 0.27) and 23.14 (± 0.30) °C for the control, low-, mid- and high-concentration groups,

respectively (Table 1.3). The average relative humidity during exposure was 48.73 (± 0.92), 49.47 (± 0.90), 46.75 (± 0.80) and 39.02 (± 0.84) % for the control, low-, mid-, and highconcentration groups, respectively.

The oxygen concentration during exposure was in the range of 20.2 – 20.4% (v/v) and the carbon dioxide concentration was in the range of 0.275 – 0.549% (v/v), which was

well within the limits of >19% oxygen and <1% carbon dioxide described in OECD guideline 412.

Conclusions:
Under the conditions of the current study, sub-acute inhalation exposure to Dimethylethylamine at actual concentrations of:
• 249.0 ppm resulted in local toxicity in the upper respiratory tract as characterized by degeneration of the respiratory and olfactory epithelium in the nasal tissues and systemic toxicity, as evidenced by significant decrease in body weight and food consumption;
• 49.7 ppm resulted in no systemic toxicity but presence of adverse local changes in the upper respiratory tract, as characterized by degeneration of the respiratory and olfactory epithelium in the nasal tissues;
• Both in the 49.7 and 249.0 ppm groups, substantial, but not full recovery of the nasal pathology was observed within a 4-week post-exposure recovery period;
• 10 ppm resulted in no systemic or local adverse changes.

Based on these findings, the No-Observed-Adverse-Effect-Concentration (NOAEC) for systemic toxicity upon sub-acute inhalation exposure to Dimethylethylamine in rats was placed at 49.7 ppm, and the NOAEC for local toxicity was placed at 10.0 ppm.
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 November 2018 - 01 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SAMP160321
- Expiration date of the lot/batch: 17 April 2019


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a refrigerator (2 to 8°C), desiccated, in the dark.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:WI(Han) strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: yes]
- Age at study initiation: 53 to 59 days
- Weight at study initiation: Males :222 to 318 g, Females: 143 to 203 g
- Fasting period before study: N/A
- Housing: Cages-Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. The cages constituting each group were blocked together by sex on separate batteries. Five of the same sex per cage (main study and recovery), unless reduced by mortality or isolation. Wood based bedding which was changed at appropriate intervals each week.
- Diet (e.g. ad libitum): Teklad 2014C Diet. Non-restricted (removed overnight before blood sampling for hematology or blood chemistry and during the period exposure).
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Non-restricted (except during exposure).
- Acclimation period: 11 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

IN-LIFE DATES (Main study): From: 08 November 2018 To: 18 to 19 February 2019
IN-LIFE DATES (Recovery study): From: 08 November 2018 To: 01 April 2019
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Route: Inhalation - snout only exposure.
Control (Group 1): Air only.
Duration of daily exposure: 6 hours. Weeks 1 to 12: 5 days each week; Week 13: 7 days.
Training for dosing: The animals on study were acclimated to the method of restraint, over a 3 day period immediately preceding the first test item exposure.

Exposure System:
Flow through nose-only chamber
Aluminum alloy construction comprising a base unit, three animal exposure sections, a top section and a pre-chamber

Animal Restraint:
Plastic nose-only restraint tube

Atmosphere Generation:
Glass sintered vaporizer
The test item was supplied to the generator, via a feed line, from a syringe driven at a constant rate by a syringe pump

Inlet Airflow:
From in-house compressed air system – breathing quality
Generator flow: 10-60 L/minute

Extract Airflow:
Drawn by in-house vacuum system
Filtered locally
Extract flow: 40-160 L/minute

Airflow Monitoring:
High quality tapered tube flowmeters - calibrated daily
In-line flowmeters monitored continuously

System Containment:
Systems housed in separate ventilated cabinets.

Administration:
Test group animals (Groups 2 – 4) were exposed to an atmosphere containing dimethylethylamine.
Group 1 animals were exposed to compressed air only
Animals were exposed on five days each week for 13 weeks. Additional animal exposures were conducted on Week/Day 13.6, 13.7 and 14.1 to cover end of study
investigations.
Duration of exposure was 6 hours each day
Exposures commenced on 19 November 2018
Different exposure levels were achieved by varying the concentration of test item in the exposure systems, whilst keeping the duration of exposure constant
The animals on study were acclimatized to the method of restraint for three consecutive days preceding their first exposure
System operating conditions were amended at the discretion of the Study Director to maintain achieved atmosphere concentrations close to target.

Concentration:
Atmosphere samples collected as follows:
Collection media: Dreschel head and solvent trap (bubbler)
Sample solvent: Methanol
Sample flow: 2.0 L/minute
Sample volume: Measured by wet-type gas meter
Sample frequency: 1 sample from Group 1/day (taken at approximately 180 minutes into exposure)
Minimum of 3 samples from Group 2, 3 and 4/day (taken at approximately 60, 180 and 300 minutes during exposure)
Sample location: Animal exposure port
Sample analysis: Chemical
During preliminary characterization trials an assessment was made of the percentage breakthrough of test item through the sample collection media; this was achieved by setting up two bubblers in series and collecting a sample of test atmosphere. The acceptable breakthrough limit to the second solvent trap is = 10%. The percentage break through the sample collection media was less than 10%, therefore one bubbler was used on study to collect chamber atmosphere samples.

Chamber air temperature was measured throughout exposure using an electronic thermometer probe placed in the breathing zone of the animals via an unused exposure port. Chamber air temperature was monitored continuously and recorded at 60-minute intervals.

Chamber relative humidity was measured throughout exposure using an electronic hygrometer probe placed in the breathing zone of the animals via an unused exposure port.
Chamber relative humidity was monitored continuously and recorded at 60-minute intervals.

The mean achieved atmosphere concentrations were 103, 100 and 106% of target for Groups 2, 3 and 4, respectively. Initially the inter and intra exposure variation was higher than anticipated. Bubbles were observed to be forming in the syringes and feed lines containing the test item. This was attributed to the test item expanding as it warmed following refrigerated storage and was
remedied by allowing the test item to warm to ambient temperature prior to generation. Subsequently the test item was observed to be vaporizing in the feed lines with an inversely
proportional relationship to target atmosphere concentration. This was considered to be a consequence of the very low feed rates required to achieve the target atmosphere concentrations and was remedied by reducing the diameter of the feed lines.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 Weeks.
Frequency of treatment:
Dimethylethylamine was administered to Wistar Han rats by snout-only inhalation exposure for 6 hours per day (5 days per week) for 13 weeks.
Dose / conc.:
0 ppm
Dose / conc.:
10 ppm (nominal)
Remarks:
Analytical value: 10.3 ppm
Dose / conc.:
30 ppm (nominal)
Remarks:
Analytical value: 29.9 ppm
Dose / conc.:
100 ppm (nominal)
Remarks:
Analytical value: 106 ppm
No. of animals per sex per dose:
10 animals, per sex, per dose + 10 animals per sex for the recovery groups (control and high dose)
Control animals:
yes
Details on study design:
The target doses used in this study (0, 10, 30 and 100 ppm) were selected based on the results of a previous 28-day inhalation study:
After 28 days of exposure to 50 and 250 ppm, degeneration of respiratory and olfactory epithelium in the nose was observed. At 250 ppm, up to moderate degeneration was observed in all animals in all nasal sections with the exception of level 1. Additionally, reduced body weight (almost 10% in males) and reduced food consumption was recorded at 250 ppm. No changes were caused by 10 ppm. Due to the severity of the corrosion of the nasal tissue and body weight differences of almost 10%, 250 ppm was considered to high for a 90-day exposure. Consequently, a high exposure level of 100 ppm was selected. Based on the results obtained at 50 ppm, local effects were still expected in the nose but it was considered that these would be tolerable. Intermediate and low exposure levels of 30 or 10 ppm were selected to assess any relationship to exposure level.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Cages were inspected daily for evidence of animal ill-health amongst the occupants. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
Signs Associated with Dosing
Daily during the first four weeks of treatment on exposure days and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Pre-exposure observation
As each animal was returned to its home cage
As late as possible in the working day

In addition observations were made in the treatment period, on days without exposures during Weeks 1 to 4, at the following times during the day:
Early in the working day (equivalent to pre-exposure observation)
As late as possible in the working day

Observations during exposure is severely restricted due to tube restraint.

Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

BODY WEIGHT: Yes
The weight of each animal was recorded twice weekly from one week before treatment commenced, on the day that treatment commenced (Day 1) and during Weeks 1 to 4. Weekly body weights were recorded during Weeks 5 to 13, during recovery and on the day of necropsy.

FOOD EFFICIENCY: Not specified

FOOD CONSUMPTION
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all animals were examined by means of a binocular indirect ophthalmoscope during pretreatment and Week 13.

HAEMATOLOGY: Yes
Blood samples were collected after overnight withdrawal of food duing week 13 from all animals.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

*Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent. Activated partial thromboplastin time (APTT) - using IL APTT reagent.

BLOOD CHEMISTRY: Yes
Blood samples were collected after overnight withdrawal of food duing week 13 from all animals.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea*
Blood urea nitrogen (BUN)
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

*Numerically equivalent to blood urea nitrogen (BUN)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Yes
Sensory reactivity and grip strength assessments were performed (on non-dosing days) on all main study animals in Groups 2 and 3 and all recovery phase animals during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.
The following measurements, reflexes and responses were recorded:

Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers).
Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded.
Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed.
Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded.
Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.

Motor Activity
During Week 12 of treatment (on non-dosing days), the motor activity of all main study animals in Groups 2 and 3 and all recovery phase animals was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.


IMMUNOLOGY: Not specified

OTHER:
Estrous Cycles – Vaginal Smears: Dry smears were taken For 14 days during Weeks 12 and 13 of treatment and during the recovery phase, using cotton swabs.

Mortality: A viability check was performed near the start and end of each working day. Animals were isolated or killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases.

All observations regarding Thyroid hormone analysis please see materials and methods section below.
Sacrifice and pathology:
All main study and recovery animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were checked before disposal of the carcass.
Other examinations:
Bone Marrow
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment or recovery periods and from animals killed prematurely during the study

Bronchoalveolar Lavage (BAL)
The right lung was used for bronchoalveolar lavage sampling and the left lung was processed for histology and light microscopy.

Sperm Analysis
Immediately after scheduled sacrifice of each male and collection of blood and bone marrow, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.
The following tests were performed:
Sperm motility – all groups
Sperm morphology – Groups 1 and 4
Sperm count – all groups
Homogenisation-resistant spermatid count – all groups

Stage-dependent Evaluation of Spermatogenesis
Stage dependent evaluation of spermatogenesis was conducted on sections of testes from all animals of Groups 1 (Control) and 4 (106 ppm) sacrificed on completion of the scheduled treatment period prepared and stained using the PAS method. A qualitative examination of spermatogenic stages was made for normal progression of the stages of the spermatogenic cycle, cell associations, and proportions expected to be present during normal spermatogenesis.



Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related clinical signs or dosing observations during the 13 weeks of treatment or during the 6 week recovery period.
Signs associated with the administration procedure included wet fur and/or red staining of the head, nose and eyes on return to home cage, in which the majority had resolved by end of working day. These signs were seen in animals from all groups including control, therefore are considered to be due to the method and duration of restraint and are commonly seen on inhalation studies of this study design. There were no test-item related effects observed during the physical examination and arena observations.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was one unscheduled death. A Group 2 male, number 20, died under anaesthetic during blood sample collection for hematology and blood chemistry during Week 13. The reason for death is unknown as the animal was considered normal prior to induction of anaesthesia and no macroscopic abnormalities were seen at necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
After thirteen weeks of treatment, group mean body weight gain was lower than control for both sexes exposed to 106 ppm (0.77X and 0.79X control, males and females respectively). Differences in body weight did not reach statistical significance.
There were no test-item related effects on body weight (gain) for either sex exposed to 10.3 or 29.9 ppm.
After 6 weeks of recovery, group mean body weight gain was higher than control for males previously exposed to 106 ppm (1.36X control), while high-dose females gained weight comparable to the controls.
Since effects were transient and body weight gain reached or exceeded the control values quickly after the end of the exposure period, this change was likely secondary to the nasal irritation / stress. It was therefor considered treatment-related but non-adverse.

Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no significant differences in food consumption after 13 weeks of treatment or 6 weeks of recovery.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was a higher incidence of superficial opacities in males that received 106 ppm after 13 weeks of treatment, evaluation of animals in groups that received 29.9 or 10.3 ppm did not reveal a similar effect. This finding is considered incidental in absence of a similar effect in females or animals in the lower exposure levels.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on haematology.
All differences from control were minor, lacked exposure relationship or were inconsistent between the sexes. Therefore, these were considered to be due to individual variation and unrelated to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on blood chemistry.
All differences from control were minor, lacked exposure relationship or were inconsistent between the sexes. Therefore, these were considered to be due to individual variation and unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects.
Group mean hindlimb grip strength was higher than control for all treated groups (not exposure related), however all were within the range of the historical data therefore this was considered incidental. A small number of differences attained statistical significance, however these were isolated and are attributed to normal variation.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects.
All differences from control were minor, lacked exposure relationship or were inconsistent between the sexes. Therefore, these were considered to be due to individual variation and unrelated to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No differences between control and treated animals were observed after 13 weeks or the additional 6 week recovery period.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Animals Killed After 13 Weeks of Treatment: Changes related to treatment with the test item were seen in the nose/turbinates. Degeneration/atrophy of the olfactory epithelium mainly affecting the dorsal parts of the nasal vestibules was observed in animals receiving 29.9 or 106 ppm and was associated with loss of axon bundles in the sub adjacent lamina propria. Incidence and severity of these changes showed an exposure level response.

Animals Killed After 6 Weeks of Recovery: Minimal degeneration/atrophy of the olfactory epithelium associated with loss of axon bundles in the sub adjacent lamina propria was observed in animals previously exposed to 106 ppm. These changes were mainly distributed in the dorsal part of the nasal cavities.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Estrus cycle: When compared with control, estrus cycles in females exposed to 106 ppm during Weeks 12 and 13 showed a higher number of irregular cycles. Of the 20 animals, 5 had irregular cycles versus 1 control animal. There were also two individuals exposed to 106 ppm that either had extended estrus or were determined to be acyclic. During the recovery period, there was one control female with an irregular cycle and extended estrus for two females previously exposed to 106 ppm. Since a similar incidence was also observed in the low dose (2 (out of 10) low dose females showed an irregular cycle and 1 additional female had an extended estrus), while no mid-dose females showerd irregularities (lack of dose-response), and because a similar incidence of divergence was also observed in one of the recent historical control studies, the changes in the high-dose were considered as incidental.

T3/T4 analysis: There were no test item-related effects. All samples taken from all groups, including control, at termination and from control and animals previously exposed to 106 ppm at the end of the recovery phase showed T3 and T4 concentrations were consistent among groups.

Thyroid Stimulating Hormone analysis: Individual serum TSH concentrations were found to be variable. Group mean TSH concentrations were lower for males exposed to 10.3 ppm when compared with control. Group mean TSH concentrations for males exposed to 29.9 or 106 ppm were similar to control. There was a slight increase in TSH concentrations for males previously exposed to 106 ppm when compared with control. Females showed an increase in TSH concentrations with increasing concentration of Dimethylethylamine when compared with control. There was a slight decrease in TSH concentrations for females previously exposed to 106 ppm when compared with control.
Given the high degree of variability, lack of exposure related response in males and inconsistency between the sexes, it is considered the observed changes are likely to be a result of biological variation rather than a test-item related effect.

Sperm Analysis: No adverse effects on sperm motility, testicular spermatid numbers, cauda epididymal sperm numbers or sperm morphology were observed following treatment with Dimethylethylamine compared with control.

Bronchoalveolar Lavage (BAL): There were no test item-related effects.
Group mean cell counts were variable when compared with control, however individual values for test animals were within the control range and therefore all differences were attributed to normal biological variation.

Total Protein and Lactate Dehydrogenase: Although group mean data may suggest lower total protein and lactate dehydrogenase concentrations in treated males and higher total protein and lactate dehydrogenase concentrations in females, there was a large degree of variation and overlap in individual data when comparing test data with control, so there is no convincing test item-related effect.
After 6 weeks of recovery, higher group mean total protein (up to 1.82X control) and lactate dehydrogenase concentrations (up to 1.64X control) were observed in both sexes exposed to 106 ppm when compared with control.
Dose descriptor:
NOAEC
Remarks:
nasal local effects
Effect level:
10.3 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
31 mg/m3
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
>= 106 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
318 mg/m3
Critical effects observed:
no

Summary of treatment related findings in the nose/turbinates for animals killed after
13 weeks of treatment
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Exposure level (ppm) 0 10.3 29.9 106 0 10.3 29.9 106
Degeneration/Atrophy, Olfactory
Epithelium with Loss of axon Bundles
               
Minimal 0 0 2 3 0 0 4 1
Slight 0 0 0 6 0 0 0 5
Moderate 0 0 0 1 0 0 0 4
Total 0 0 2 10 0 0 4 10
Number of tissues examined 10 10 10 10 10 10 10 10

Summary of treatment related findings in the nose/turbinates for animals killed after
6 weeks of recovery
Group/sex 1M 4M 1F 4F
Exposure level (ppm) 0 106 0 106
Degeneration/Atrophy, Olfactory
Epithelium with Loss of axon Bundles
       
Minimal 0 5 0 4
Slight 0 3 0 0
Total 0 8 0 4
Number of tissues examined 10 10 10 10
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
A detailled read across justification has been attached to IUCLID chapter 13.
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
>= 106 ppm
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
10.3 ppm
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
500 mg/m³
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: inhalation
Remarks:
combined repeated dose and reproduction/developmental tox screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 2018 - Aug 2019
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)
Version / remarks:
Jul 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
- Sponsor: BASF SE, Ludwigshafen, Germany
- Purity: 99.9%
- Storage conditions: Room temperature
- Homogeneity: Given
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:Charles River Laboratories, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation:males (10-11 weeks old) and females (9 weeks old)
- Weight at study initiation: (P) Males: 380 g; Females: 218 g;
- Housing:
From delivery until randomization, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECNIPLAST, Hohenpeißenberg, Germany. From randomization onwards, the rats were housed individually in Polycarbonate cages type IIIsupplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel,Germany, with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
• Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
• During exposure, male and female rats (until gestation) were housed in wire cages, type. DK III supplied by Becker & Co., Castrop-Rauxel, Germany, and female rats (from PND4 onwards) in perforated polycarbonate cages type III. “Exposure systems”.Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation. For enrichment wooden gnawing blocks (Typ Lignocel® block large, J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany) were added. In addition, in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX B.V., Elst, Netherland were added.

- Diet: ad libitum; Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%):45-65%
- Air changes (per hr):15 times per hour.
- Photoperiod (hrs dark / hrs light):12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Whole-body exposure systems
- Method of holding animals in test chamber: The animals were kept single in wire cages located in a glass-steel inhalation chamber.
- Source and rate of air: 1.1 m³
- Method of conditioning air: The animals were housed in fully air-conditioned rooms in which central air conditioning
- Temperature, humidity in air chamber: temperature of ~22°C and relative humidity of ~50%.
- System of generating particulates/aerosols:
• Piston metering pumps KP 2000 (DESAGA; SARSTED AG & Co, Nürnbrecht, Germany)
• Atomization vaporizers (glass) with thermostat (BASF SE, Ludwigshafen, Germany)
• Thermostat (JULABO Labortechnik GmbH, Seelbach, Germany)
The test substance was used unchanged. For each concentration, the test substance was supplied to the two-component atomizer of a thermostated vaporizer at a constant rate by means of the metering pump. The vapour/ air mixture was generated by spraying the substance with compressed air into a counter current of conditioned supply air (about 50% ± 20% relative humidity, 22°C ± 4°C). Thereafter it was further mixed with conditioned supply air and passed into the inhalation system.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Calculation of nominal concentrations
The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations.

Analytical determination of concentrations
Principle: The concentrations of the test substances in the atmospheres were determined by calibrated on-line gas chromatography method (GC) using on-line microGC.
To calibrate the microGC, an appropriate amount of test substance were weighed into gas sampling tubes with defined volume. The gas sampling tube is tempered that the test substance is evaporated completely within the tube. The so prepared atmospheres are sampled by microGC. A calibration line was formed by the pair of substance concentration and peak area. The method authorized by the contributing scientist described in detail the calibration process, the calibration curve as well as accuracy and reproducibility of the method. The non-GLP raw data of methods description were archived with the study raw data.
During the study, atmospheres were sampled by microGC via a stream selector, which switch through all concentration groups including the control group. Between the measurement of each group, an appropriate flush time was included to avoid carry over effects. The measured data were transferred to BaseLab, which generated hourly means for each test group. Daily means and standard deviations were calculated based on hourly means of each concentration for each exposure.
Duration of treatment / exposure:
Groups of 10 male and 10 female Wistar rats (F0 animals) per test group were exposed in a whole-body inhalation system to dynamic atmosphere of the test substance for 6 hours per day on each day.The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 6 days postmating in males, and the gestation (up to GD 19) and the lactation period in females from PND 4 onwards up to the day of scheduled sacrifice of the animals. The target concentrations were 125 mg/m³ (34 ppm1), 500 mg/m³ and 1500 mg/m³). A concurrent control group was exposed to conditioned air. For adaptation to the exposure conditions the animals were placed into exposure cages before start of the exposure period (pre-exposure period) on study days -5, -4 and -1 (details are available in the raw data). This pre-exposure was performed in the animal room.

Duration of the exposure:
Males:
a) 14 days premating
b) up to 14 days mating
c) sacrifice after a minimum of 28 days after the first exposure
Females:
a) 14 days premating
b) up to 14 days mating
c) during the pregnancy up to and including GD 19
d) after lactation from PND 4 onwards until one day before necropsy

The animals did not have access to water or feed during the exposure.

For adaptation to the exposure conditions the animals were placed into exposure cages before start of the exposure period (pre-exposure period) on study days -5, -4 and -1. This pre-exposure was performed in the animal room.
Frequency of treatment:
6 hours/ day, every day
Dose / conc.:
125 mg/m³ air (nominal)
Remarks:
123 mg/m³ analytical
Dose / conc.:
500 mg/m³ air (nominal)
Remarks:
505 mg/m³ analytical
Dose / conc.:
1 500 mg/m³ air (nominal)
Remarks:
1513 mg/m³ analytical
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
The animals were delivered and subjected immediately to the acclimatization period in which they were adapted to the surroundings.
Prior to the pre-exposure period, the animals were distributed according to body weight among the individual test groups, separated by sex. The weight variation of the animals used did not exceed ± 20 percent of the mean weight of each sex. The list of randomization instructions was compiled with a computer.
For each neurofunctional test and motor activity measurement, separate randomization lists were created. The list of randomization instructions was compiled with a computer (Laboratory data processing, Experimental Toxicology and Ecology, BASF SE).
All animals were exposed to the respective concentrations of test substance for 6 hours a day according to the time schedule (exception: no exposure on the day of FOB/MA). Control animals were exposed to conditioned air. All animals were observed daily for any clinical signs during the study period.
A detailed clinical observation (DCO) was performed in all animals once before the first exposure and weekly thereafter.
Males and females from the same test group were mated, after two weeks of treatment, overnight at a ratio of 1:1.
The females were allowed to deliver and rear their pups until PND 4 (standardization) or PND 13.
After the standardization of the litters, the parental female animals were exposed to the respective concentrations of test substance for 6 hours a day until one day before scheduled sacrifice.
Blood samples were taken from surplus pups at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia for hormone measurement.
On study day 30, a functional observational battery and motor activity measurement were carried out in five male animals per group.
On study day 57, a functional observational battery and motor activity measurement were carried out in five female animals (with litter) per group.
Blood samples were taken from all F0 parental male animals of all test groups shortly before sacrifice and from all F0 parental female animals of all test groups on PND 14.
The male and female animals were sacrificed 34 and 59 days, respectively, after the beginning of the treatment, and examined.

Standardization of litters (F1 generation pups)
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Surplus pups were sacrificed. Standardization of litters was not performed in litters ≤ 8 pups.

Pups after standardization
On PND 4, as a result of standardization, the surplus pups or 2 preferably female pups per litter, respectively, were sacrificed under isoflurane anesthesia by decapitation. Blood were sampled for determination of thyroid hormone concentrations. All pups were examined externally and eviscerated; their organs were assessed macroscopically.

Pairing of F0 generation parental animals
In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 6.30 - 9.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1".

Observations and examinations performed and frequency:
CLINICAL EXAMINATIONS
Parental animals
Mortality:
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the BASF SE Laboratory for Pathology, Experimental Toxicology and Ecology, Ludwigshafen, Germany.
Clinical observations:
The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days. During exposure, a group wise examination only was possible. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings. The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.
Food consumption:
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female F0 animals).
• Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0-7, 7-14, and 14-20.
• Food consumption of F0 females which gave birth to a litter was determined on PND 1-4, 4-7, 7-10 and 10-13.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.
Body weight data:
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results. The following exceptions are notable for the female animals:
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day after parturition (PND 1) and on PND 4, 7, 10 and 13.
Females without litter or waiting for necropsy, were weighed weekly. The body weight data of these individuals are not reported in the Summary but in the Individual Tables (PART II).
Detailed clinical observations:
Detailed clinical observations (DCO) were performed in all animals once prior to the first treatment (day 0) and at weekly intervals during the exposure period. The examinations startedin the morning. The findings were ranked according to the degree of severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed: Abnormal behavior in “handling”, Fur, Skin, Posture, Salivation, Respiration, Activity/arousal level, Tremors, Convulsions, Abnormal movements, Gait abnormalities, Lacrimation, Palpebral closure, Exophthalmos (Protruding eyeball), Assessment of the feces excreted during the examination (appearance/consistency), Assessment of the urine excreted during the examination, Pupil size
Functional observation battery:
A functional observation battery (FOB) was performed in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. There is no exposure of the concerning animals as well as the other 5 animals of the same test group. At least one hour before the start of the FOB the animals were transferred singly to Polycarbonate cages (floor area about 800 cm²). The cages were placed in the racks in a randomized order (randomization based upon animal’s number). Drinking water was provided ad libitum whereas no food was offered during the measurements. The FOB started with passive observations, without disturbing the animals, followed by removal from home cage, and open field observations in a standard arena. Thereafter, sensorimotor tests and reflex tests were conducted. The examinations were carried out by trained technicians which performed positive control studies as part of their training. Another technician documented all findings and values obtained. The findings were ranked according to the degree of severity, if applicable.
Home cage observations:
The animals were observed in their closed home cages; any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to: Posture, Tremors, Convulsions, Abnormal movements, Gait, Other findings
Open field observations:
The animals were transferred to a standard arena (50 x 50 cm with sides of 25 cm high) and observed for at least 2 minutes. Following parameters were examined: Behavior on removal from the cage, Fur, Skin, Salivation, Nasal dischare, Lacrimation, Eyes/pupil size, Posture, Palpebral closure, Respiration, Tremors, Convulsions, Abnormal movements/ stereotypies, Gait, Activity/arousal level, Feces (appearance/consistency) within 2 minutes, Urine (amount/color) within 2 minutes, Rearings within 2 minutes, Other findings
Sensory motor tests/reflexes:
The animals were removed from the open field and subjected to following sensorimotor or reflex tests: Reaction to an object being moved towards the face (Approach response), Touch sensitivity (Touch response, Vision (visual placing response), Pupillary reflex, Pinna reflex, Audition (Startle response), Coordination of movements (Righting response), Behavior during handling, Vocalization, Pain perception (Tail pinch), Other findings, Grip strength of forelimbs, Grip strength of hindlimbs, Landing foot-splay test
Motor activity measurement:
The Measurement of motor activity (MA) was measured at the end of the exposure period in the first 5 surviving parental males and the first 5 surviving females with litter (in order of delivery) per group. Motor activity was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.
Estrous cycle determinations:
For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization (the estrous cycle data of these individuals were not be reported and can be found in the raw data). For a minimum of 2 weeks prior to mating estrous cyclellength was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.
Male reproduction data:
The pairing partners, the number of mating days until vaginal sperm was detected in the female animals, and the gestational status of the females were recorded for F0 breeding pairs. For the males, mating and fertility indices were calculated for F1 litters according the formulas given in OECD TG 422 (2016).
Female reproduction and delivery data:
The pairing partners, the number of mating days until vaginal sperm was detected and gestational status was recorded for F0 females. For the females, mating, fertility and gestation indices were calculated for F1 litters according to the formulas given in OECD TG 422 (2016).
The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula (given in OECD TG 422 (2016)): Live birth index (%) = (number of liveborn pups at birth / total number of pups born) * 100.
The implantations were counted and the postimplantation loss (in %) was calculated according to the following formula (given in OECD TG 422 (2016)): Postimplantation loss (%) = ((number of implantations - number of pups delivered) / number of implantations) * 100.

CLINICAL PATHOLOGY
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The parameters listed below were examined in the first 5 surviving parental males per group at termination and in the first 5 females with litters (in order of delivery) per group at PND 14.
Hematology:
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), differential blood count, Reticulocytes (RET):
Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. (reference: Hematology: Principles and Procedures, 6th Edition, Brown AB, Lea & Febiger, Philadelphia, 1993, page 101). Only evaluated blood smears were archived.
Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany). Measured parameter was prothrombin time.

Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the following clinicochemical parameters: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Na, K, Cl, inorganic Phosphate (INP), Ca, Urea, Creatinine, Glucose, total Bilirubin, total protein, Albumin, Globulins, Triglycerides, Cholesterol, Bile acids.
Thyroid hormones
Blood samples were taken from all surplus pups per litter at PND 4 as well as one male and one female pup per litter at PND 13 by decapitation under isoflurane anesthesia. Additionally, blood samples from all dams at PND 14 and all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. The adults were fastened before the blood sampling. All generated serum samples were frozen at -80°C until measurement.
Blood samples from the adult males and the PND 13 pups were assessed for serum levels for thyroid hormones (T4 and TSH). (For technical reasons the values of PND 13 pups (males Nos. 201-240, females Nos. 301-340) were listed in the mean and individual tables under test groups 10, 11, 12 and 13 instead of 0,1, 2 and 3). The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits and a Gamma-Counter (LB 2111, Berthold, Germany). T4 Elisa was measured with a Sunrise MTP-reader, Tecan AG, Maennedorf, Switzerland, and evaluated with the Magellan-Software of the instrument producer.
Sacrifice and pathology:
Necropsy:
All parental animals were sacrificed under pentobarbitone anesthesia. The left and right brachial vessels were opened, by deep cuts through the pectoral muscles along both sides of the rib cage. Caution was exercised to avoid destruction of the axillary lymph nodes. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
Organ weights
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals (terminal body weight), Epididymides, Ovaries, Prostate (ventral and dorsolateral part together, fixed), Seminal vesicles with coagulating glands (fixed), Testes, Thyroid glands (with parathyroid glands) (fixed), Uterus (with cervix)
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations): Adrenal glands (fixed), Brain, Heart, Kidneys, Liver, Lungs, Spleen, Thymus (fixed).
All paired organs were weighed together (left and right).

Organ/tissue fixation
The following organs or tissues of all parental animals were fixed in in 4% neutral-buffered formaldehyde or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Esophagus, Epididymides (modified Davidson’s solution), Extraorbital lacrimal glands, Eyes with optic nerve (modified Davidson’s solution), Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus (uteri of all apparently nonpregnant animals or empty uterus horns were stained according to Salewski E, 1964), Vagina.
Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings. For further details on histopathological examination please see attached background material.
Special attention was given to the stages of spermatogenesis and histopathology of interstitial testicular cell structure.
The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
A correlation between gross lesions and histopathological findings was attempted.
Whenever in the ovary the diagnosis: „no abnormalities detected” was used that implies that all different stages of functional bodies (especially corpora lutea) were present and normal.
Peer review
After completion of the histopathological assessment by the study pathologist an internal peer review was performed including prostate glands and ovaries of all animals of all test groups. Results presented in this report reflect the consensus opinion of the study pathologist and the peer review pathologist.
Statistics:
Food consumption, body weight (bw) and bw change (parental animals and pups; for pup weights, litter means were used), gestation days, anogenital distance, anogenital index: Simultaneous comparison of all dose groups with control group (CoG) using DUNNETT test (2sided)
Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups: Pair-wise comparison of each dose group with CoG using FISHER'S EXACT test (onesided)
Mating days until day 0 p.c., %postimplantation loss, pups stillborn, %perinatal loss, nipple development, Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival index:Pair-wise comparison of the dose group with CoG using WILCOXON test (onesided) with BONFERRONI-HOLM adjustment
%live male day x, %live female day x: WILCOXON test (2sided)
Number of cycles and Cycle Length, Rearing, grip strength of fore limbs and hind limbs, landing foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of the dose groups with CoG was performed using WILCOXON test (2sided) for the equal medians.
Blood parameters: Parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with CoG was performed using WILCOXON-test (2sided). For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided).
Weight parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with CoG was performed using WILCOXON-test (2sided)for the equal medians.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During premating and mating periods, all parental male and female animals (10 out of 10) of test group 3 (1500 mg/m³) showed salivation, piloerection and reduced attention during exposure. Furthermore, a discolored fur (reddish) occurred in one female of test group 3 during premating and in each two males and females of test group 3 during mating. Salivation, piloerection and reduced attention lasted up to scheduled sacrifice (during postmating period) in all male animals of test group 3. These clinical signs were also seen in all test group 3 female animals during gestation period. Furthermore, a discolored fur (reddish) occurred in six females of test group 3 during gestation. During lactation period, all female animals of test group 3 had piloerection and their attention was reduced during exposure.
The above-mentioned clinical findings in test group 3 male and female animals were assessed as treatment-related and adverse.
No clinical signs or changes of general behavior which may be attributed to the test substance, were detected in any male or female F0 parental animals at concentrations of 125 or 500 mg/m³ during premating, mating, gestation, lactation and postmating periods. For one female animal of test group 2 (No. 127 - 500 mg/m³) hypothermia and piloerection were recorded at the end of the gestation period (GD 23-24). In the litter of this female, all pups were stillborn (PND 0). Furthermore, test group 3 female animal No. 133 (1500 mg/m³) had an opacity on its right eye during PND 15-18. These single events were assessed to be incidental and not related to treatment.
One sperm-positive female of the control group (No. 104) did not deliver F1 pups.

Detailed clinical observations (DCO)
No test substance-related findings were observed in male and female animals of all test groups (125, 500 or 1500 mg/m³).
One spontaneous finding occurred in test group 3 during DCO on day 56, i.e. an opacity on the right eye of female animal No. 133.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any of the groups.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
During premating period, test group 3 F0 male animals (1500 mg/m³) lost weight at all time points. Thus, the mean body weight (change) was statistically significantly reduced during thewhole premating period. During premating, mating and postmating, their mean body weight was constantly decreased (up to -9%) compared to the concurrent control.
The mean body weights of the test group 3 F0 parental female animals (1500 mg/m³) were statistically significantly decreased compared to the concurrent control values during gestation (GD 7-20: up to -9%) and lactation (PND 7-13: up to -12%). In test group 3, the mean body weight change of the F0 females was statistically significantly reduced during premating (0-13) showing a body weight loss, during gestation (GD 0-20: -21% below control) and lactation (PND 1-13: -52% in comparison to the concurrent control). The body weight decrease of the test group 3 male and female parental animals (1500 mg/m³) was assessed as treatment-related and adverse.
In test group 2 (500 mg/m³), F0 parental males gained less weight during premating (attaining statistical significance) but recovered afterwards during mating. Since the body weight gain was not affected during mating and postmating, this marginal decrease during one short time period was not assessed as treatment-related and adverse.
The mean body weight (change) of the test group 1 males and females and test group 2 females were comparable to the concurrent control group during the entire study period.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption of the F0 parental males of test group 3 (1500 mg/m³) was statistically significantly reduced during premating period(up to 24% below concurrent control). If calculated for the whole premating period (days 0-13), these males consumed approx. 23% less food than the controls.
Consistently to the males, food consumption of the F0 parental test group 3 females (1500 mg/m³) was statistically significantly reduced during premating (up to -20%; days 0-13: approx. 17% below control), gestation (GD 0-20: -13%) and lactation (PND 4-13: up to -23%; PND 1-13: approx. 17% below control).
The reduction in food consumption of the test group 3 males and females in all treatment periods during lactation was assessed as treatment-related and adverse.
In test group 2 (500 mg/m³), the food consumption of thetest group 2 females was generally comparable to control during premating and gestation. During lactation, food consumption of the F0 females was reduced compared to control from PND 4 onwards (PND 4-7: -10%, without statistical significance; PND 7-13: -14%, with statistical significance).
For the test group 2 females, the reduction in food consumption during lactation was assessed as treatment-related but not as adverse since it caused neither a corresponding reduction of body weight/body weight gain nor any other measurable effect on the well-being of those animals.
Food consumption of the F0 male and female animals in test group 1 (125 mg/m³) and of the F0 males in test group 2 (500 mg/m³) was generally comparable to the respective control group during the entire treatment period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
At the end of the administration period, in parental males of test group 1 (125 mg/m3) relative neutrophil counts were significantly decreased, but this alteration was not concentration dependent In dams at PND14, absolute reticulocyte counts were significantly lower compared to study controls. However, this change was also not concentration-dependent. Therefore, these alterations were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the administration period, in parental males and dams at PND14 of test group 3 (1500 mg/m3) bilirubin was slightly increased above historical controls values in both sexes, but attained statistical significance in females only. Stress as well as fasting lead to an increase in serum bilirubin. Food consumption (and body weight) of high dose animals was severely reduced, adrenal gland weights were reduced as a result of stress (all likely caused by the local corrosive effects in the nose and larynx). Consequently, the slight change in bilirubin is not considered to be a direct effect of treatment, but secondary to stress and reduced food consumption.
Values for bile acids were also slightly above historical control values, though differences were not statistically significant. In females, no dose response was observed as the highest value occured in low dose animals. For males, only 2 of 5 animals had serum bile values just above the historical control data (though several values in the same range occured in the historical controls, but were marked as outliers). Because of the small increase, no statistical significance and no dose repsonse in females, the change was not considered to be related to treatment.
In parental males of test group 3 (1500 mg/m3) alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were significantly increased. In males of test groups 2 and 3(500 and 1500 mg/m3) chloride levels were significantly decreased whereas in males of test groups 1, 2 and 3 (125, 500 and 1500 mg/m3) inorganic phosphate levels wer esignificantly higher compared to study controls. However, all mentioned parameter values were within historical control ranges (males, ALT 0.55-0.90 μkat/L; AST 1.31-2.11 μkat/L; chloride 97.9-104.1 mmol/L; inorganic phosphate 1.45-2.06 mmol/L). Therefore, the mentioned alterations were regarded as incidental and not treatment-related.
In females of test group 3 (1500 mg/m3) potassium levels were significantly increased. The mean was above the historical control range (females, potassium 4.23-4.90 mmol/L). This was the only changed electrolyte among these individuals. Therefore, this change is regarded as treatment-related, but non-adverse (ECETOC Technical Report No 85, 2002).
In parental males (test groups 1, 2 and 3; 125, 500 and 1500 mg/m3) and in male and female pups at PND13 (test groups 11, 12 and 13; 125, 500 and 1500 mg/m3), no treatment-related alterations of T4 and TSH levels were observed.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observational battery:
No test substance-related findings were observed in male and female animals of all test groups during the home cage observation.
One male of the mid-concentration group (No. 25 - 500 mg/m³) showed manege movements during the home cage observation. Since only one animal showed this finding, it was not assessed as treatment-related and adverse.

Open field observations:
One male of the high concentration group, two males, each, of the mid- and low-concentration groups and one male of the control showed slight resistance against handling. Furthermore, two females of the mid-concentration and one female of the low-concentration group showed the same finding during open field observation. Since there was no relation to concentration, it was assessed as spontaneous. The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.

Sensorimotor tests/reflexes:
One male animal, each, of test groups 1 and 3 showed very frequent vocalizations when touched. One test group 3 male had a weak or retarded reaction to the stimulus during the pain perception test. These findingswere not assessed as treatment-related and adverse since they occurred only in individual animals and all males showed neither clinical signs nor changes in associated parameters in this study.
There were no testsubstance-related findings in male and female animals of all test groups.

Motor activity measurement:
No statistically significant changes on motor activity data (summation of all intervals) was observed in the male and female animals of all concentration groups in comparison to the concurrent control group. The animals showed a normal habituation to the test environment. Motor activity measurement (single value) was statistically significantly above the concurrent control value in males of test group 3 (1500 mg/m³) during interval 3 (513.8 vs. 236.4 in control). Since the increase was a single, isolated event, it was assessed as incidental and not treatment related.

Quantitative Parameters:
No test substance-related impaired parameters were observed in male and female animals of all test groups. The statistically significantly increased number of rearing in the females of test group 1 was assessed as incidental since there was no relation to concentration.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The significantly increased absolute and relative weights of adrenal glands in females of test group 3 were regarded as treatment related, but non-adverse. Absolute adrenal gland weights were at the upper end of historical control values whereas relative adrenal gland weights were slightly above the range of historical controls. Since the animals showed a significantly reduced terminal body weight, the finding was regarded as a secondary, stress-related response without any histopathological correlate.
The significantly decreased absolute heart weight in males and females of test group 3 as well as the significantly increased relative weights of brain, epididymides and testes in males of test group 3 were related to the significantly decreased terminal body weight in these animals.
The significantly decreased absolute weights of prostate glands in test groups 2 and 3 males, the significantly decreased weights of seminal vesicles (absolute: test groups 1 and 2, relative: test group 2,) the significantly increased absolute and relative uterus weights in test group 2 females and the significantly increased liver weights in males of test groups 1 and 2 were regarded to be incidental, as there was no concentration-response-relationship and no histopathological findings, that would explain the organ weight changes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings were observed in the nasal cavity (all four levels) of test groups 2 and 3 males and females, in the larynx (level I) of test group 3 males and females.

The nasal cavity (level I-IV) showed a degeneration/regeneration of the olfactory epithelium, characterized by one or more of the following findings: increased intercellular spaces, irregular epithelial architecture, necrotic epithelium, and an increased nuclear/ cytoplasmic ratio. Multifocally, a formation of glandular-like structures within the olfactory epithelial layer was observed. The degeneration/regeneration was mainly located in the dorsal part of the nasal septum and in the ethmoid turbinate. The severity varied from minimal to marked and was concentration-related increased.

Almost all male and female animals of test group 3 showed a minimal (grade 1) to marked (grade 4) degeneration/regeneration of the olfactory epithelium at all four levels. Few male and female animals of test group 2 showed a minimal degeneration/regeneration of the olfactory epithelium in levels II and III of the nasal cavity. The degeneration/regeneration of the olfactory epithelium was regarded as treatment-related.
Level I of the larynx showed an increased incidence of minimal epithelial alteration in males and females of test group 3. The increased incidence of epithelial alteration was regarded as treatment-related.

In the prostate of test group 3 males, a slightly increased incidence and severity of inflammation was observed. The inflammation was mainly located in the ventral part of the prostate gland and did not differ morphologically from the inflammation occurring in the control animal. Prostatic inflammation is a common background finding in rats (Creasy et al., 2012) and the incidence of this finding in the current study was rather low despite the slight increase in test group 3 animals. Therefore, and since there were no additional pathologic findings at the male reproductive tract nor any hints regarding a reduced fertility, a relation to treatment was regarded as unlikely. The slightly increased incidence of eosinophilic droplets in the kidneys in male animals of test group 3 (4/5 in test group 3 vs. 2/5 in control) was regarded as incidental, since eosinophilic droplets belong to the very common background findings in male rats (McInnes, 2012), that reach up to 100% in the historical controls. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The stages of spermatogenesis in the testes of males of the test group 3 were comparable to those of the controls. In test group 3 females the different stages of functional bodies in the ovaries were present and comparable to the control animals.

Fertility
The female animal (No. 104), which was not pregnant, did not show relevant histopathological findings of the reproductive organs. All other organs were not examined histopathologically. The male mating partner (No. 4) did not show relevant histopathological findings.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No treatment-related alterations of T4 and TSH levels were observed.
Key result
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
125 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
500 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical signs
food consumption and compound intake
Critical effects observed:
no
Conclusions:
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, the NOAEC (no observed adverse effect concentration) for local toxicity of Dimethyl(propyl)amine in the respiratory tract was the lowest tested concentration of 125 mg/m³ based on adverse effects such as degeneration/regeneration of the olfactory epithelium in the nasal cavity of the parental animals after inhalation (vapor) exposure to 500 and 1500 mg/m³.
The NOAEC for systemic toxicity was the mid concentration of 500 mg/m³ as adverse clinical signs, a reduction in food consumption together with a decrease in body weight (change) occurred in both sexes at the highest concentration of 1500 mg/m³.
Endpoint:
repeated dose toxicity: inhalation, other
Remarks:
Range finder
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Dec 2017 - Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
no guideline followed
Principles of method if other than guideline:
7-day DRF to select the concentration levels for the subacute main study.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source: Arkema, La Chambre, France
- Batch No.of test material: SAMP160321
- Expiration date: 20 Nov 2018
- Purity: 99.41%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature (in a dry, cool and well-ventilated place, storage in hermetically closed bottle at +4 °C)
- Stability under test conditions: not specified
- Solubility and stability of the test substance in water: completely soluble
- Reactivity of the test substance with water: exothermic reaction

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added:
- other information:
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
Rats were chosen, because this species is normally used in toxicity studies and is accepted by the relevant regulatory authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 251 g (males), 166 g (females)
- Fasting period: no
- Housing: single (during exposure), goup of five, seperated by sex (non-exposure)
- Diet: ad libitum
- Water: ad libitum:
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY:
- Food: Analyzed for nutrients and contaminants, including certificate
- Water: Checked routinely (physical, chemical, and microbial examination), including results

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 10 Jan 2018 (first exposure, day 0) To: 17 Jan 2018 (day 7)
Route of administration:
inhalation: mist
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: inhalation chambers consisting of a cylindrical aluminium column, surrounded by a transparent cylinder (a modification of the chamber made by ADG Developments Ltd., Codicote, Hitchin, Her ts, SG4 8UB, United Kingdom)
- Method of holding animals in test chamber: animals were secured in plastic animal holders (Battelle)
- System of generating particulates/aerosols: To generate the test atmospheres, a continuous flow of liquid test material3, controlled by a peristaltic pump (Minipulse 3, Gilson, Velliers le Bel, France) was allowed to evaporate in a mass flow controlled (Bronkhorst Hi Tec, Ruurlo, the Netherlands) stream of compressed dry air, by directing it through a glass evaporator which was kept at a constant temperature of 48˚C by circulating heated water.
- Temperature, humidity, pressure in air chamber: 22 +/- 3 °C, 30-70%, positive pressure in the central column and a slightly negative pressure in the outer cylinder
- Air flow rate: 1 liter/min

TEST ATMOSPHERE
- Brief description of analytical method used: Total carbon analysis
- Samples taken from breathing zone: yes. Empty ports were used for test atmosphere sampling
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the first exposure, calibration was performed.
10 ppm: On 21 and 22 December 2017, concentrations of 7.8, 8.5, 9.2, 9.7, 9.8, 9.8, 9.9, 10.9, 11.5, 19.7 and 21.5 ppm were analyzed
100 ppm: On 20 December 2017, concentrations of 73, 79, 109, 111, 124 and 127 ppm were analyzed
500 ppm: On 9 January 2018, concentrations of 358, 374, 440, 474, 615 and 727 ppm were analyzed
1000 ppm: On 19 December 2017, concentrations of 852, 1005, 1079, 1142, 1281 and 1338 ppm were analyzed
Duration of treatment / exposure:
7 d (5 exposure days in total)
Frequency of treatment:
6 h/d, 5 d/w
Dose / conc.:
10 ppm (nominal)
Remarks:
10.0 (± 0.1) ppm (analytical)
Dose / conc.:
100 ppm (nominal)
Remarks:
100.5 (± 0.9) ppm (analytical)
Dose / conc.:
500 ppm (nominal)
Remarks:
497.2 (± 6.5) ppm (analytical)
Dose / conc.:
1 000 ppm (nominal)
Remarks:
998.1 (± 4.3) ppm (analytical)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Selection of target concentrations for the concentration range finding study were based on the 4h LC50 that was found to be ca. 3400 ppm; no mortality was observed at a lower concentration of 2300 ppm. Although no repeated-dose inhalation toxicity data were available for Dimethylethylamine, there were data for the toxicity of the comparable substance Trimethylamine (CAS no. 75-50-3). The 4h LC50 was comparable at ca. 2400-3500 ppm. In a subacute study, mild toxicity was observed at the low concentration of 75 ppm, while mild toxicity was observed at a concentration as low as 10 ppm in a chronic study (all information available at http://www.echa.eu); thus, to enable identification of a NOAEC and characterize the concentration-response relationship, a broad concentration range was chosen for this range finding study.
Positive control:
not included
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily in the morning, halfway through exposure, and afternoon

DETAILED CLINICAL OBSERVATIONS: Yes, if necessary
- Time schedule: daily, afternoon

BODY WEIGHT: Yes
- Time schedule for examinations: 2 days before start of exposure, prior to first exposure (day 0), on day 4, and on scheduled sacrifice

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Time schedule: Once weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, adrenals, heart, kidneys, liver, lungs (with trachea), spleen, testes, ovaries.
HISTOPATHOLOGY: Yes, larynx, kidneys, nasal turbinates, tracheobronchial lymph nodes, trachea, lungs
Statistics:
- Ancova/Anova & Dunnett (or Kruskal-Wallis & Dunnett on Ranks): Pretreatment body weight, body weight after initiation of treatment, clinical pathology (hematology, clinical chemistry), bronchoalveolar lavage parameters, organ weights, sperm motility (numerical), sperm count, testicular sperm count
- No statistics were applied (only one cage/sex/group): food consumption
- Kruskal-Wallis & Wilcoxon: Estrus cyclicity: mean length of the longest cycle, number of complete cycles in the test period; sperm motility (expressed as %), sperm morphology
- Chi-Squared & Fisher’s Exact: Incidences of histopathological changes; Estrus cyclicity: number of acyclic females, number of animals with prolonged estrus period
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 ppm: all males and most females displayed dyspnea, sniffing, irregular respiration, hunched posture, ataxia, blepharospasm and piloerection and hypoactivity. Mouth breathing observed in 4/5 males and 1/5 females. Most animals displayed salivation, some grunting, chewing movement and shallow breathing.
500 ppm: dyspnea, shallow breathing, hunched posture and piloerection were noted in about half of males after first exposure
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
1000 ppm: Concentration-related substantial body weight loss in males (16% average loss of body weight between days 0 and 7). In females, statistically significant reduced growth
500 ppm: Reduced growth in males
100 ppm: Slightly reduced body weight growth in males
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1000 ppm: Decreased food intake in males and females
500 ppm: Decreased food intake in males
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
1000 ppm: Relative weights of lungs and heart statistically significantly increased in males and females. In males, relative testes and adrenals weights increased and relative liver weights decreased
500 ppm: In males, relative testes weights increased
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 ppm: Lungs of 4/5 males showed red spots. Small thymus was found in 2/5 males and 2/5 females. A small thymus found in 2/5 males and females
Some single animals in the other treatment groups showed red spots in the lungs that probably were related to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1000 ppm: Level 2 was most affected with the highest score grades for (granulocytic) inflammation, ulceration and necrosis. The damaged areas were mainly the nasal septum, the conchae and the lateral walls of the nasal cavity. In severe cases of necrosis, parts of the conchae and/or nasal septum were completely gone. High score grades for inflammation and ulceration were also observed in levels 3 and 4, while slightly lower grades were scored in levels 5 and 6. The remains of the damaged epithelium showed large areas with squamous metaplasia. In the necrotic areas sometimes colonies of (most likely) opportunistic bacteria were seen. Larynx: Minimal to mild granulocytic inflammation in 8/10, mild squamous metaplasia in 8/10 and minimal to mild ulceration in 4/10 animals. Trachea/bronchi: minimal to mild granulocytic inflammation in 9/10 animals. Lungs: alveolar hemorrhages in 4/10 animals, increased bronchus associated lymphoid tissue in 4/10, granulocytic inflammation of bronchi in 8/10, and a granulomatous inflammation in 2/10 animals.
500 ppm: olfactory epithelial atrophy in most animals. inflammatory changes were seen in several animals, in levels 1 to 4, occasionally accompanied by necrosis or ulceration. Minimal to mild granulocytic inflammation in 4/10 animals. Minimal to mild granulocytic inflammation in 3/10 animals.
100 ppm: Minimal to mild atrophy of the olfactory epithelium in levels 3, 4 and 5, mainly in the dorsal meatus.
Histopathological findings: neoplastic:
not examined
Details on results:
Under the conditions of this 7-day concentration range-finding study, inhalation exposure to the test substance at target concentrations of 10, 100, 500 and 1000 ppm resulted in the following treatment-related changes:
• At 1000 ppm, marked toxicity was observed; in nearly all animals, dyspnea, sniffing, irregular respiration, hunched posture, ataxia, blepharospasm, piloerection and hypoactivity were observed. Body weight of males exposed to the top concentration were 22% lower than controls (on day 7); for females the reduction in body weight was 15%. Relative weight of the heart and lungs was increased in male and female animals. When assessing histopathology, severe olfactory epithelial atrophy was observed in the nasal tissues. Milder effects were observed in the larynx, trachea/bronchi and lungs, mainly of inflammatory nature.
• At 500 ppm, transient clinical abnormalities (dyspnea, shallow breathing, hunched posture and piloerection) were observed in males especially. Minimal to mild atrophy of the nasal olfactory epithelium was observed in most animals when assessing nose tissue levels 3 and 4 (out of the 6 levels assessed).
• At 100 ppm, minimal atrophy of the nasal olfactory epithelium was still observed in most animals, with 2 cases of mild atrophy observed at a single level of the nose in 2 animals.
• At 10 ppm, no exposure-related toxicity was seen. Thus, the No-Observed-Adverse-Effect- Concentration (NOAEC) in this range finding study was placed at 10 ppm (target concentration).
Dose descriptor:
NOAEC
Effect level:
10 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
histopathology: non-neoplastic
Critical effects observed:
no

Table 1: Clinical observations in males

Observation Type: All Types Sex: Male

From Day -2 (Start Date) to 7 (Start Date)

0 ppm

10

ppm

100

ppm

500

ppm

1000

ppm

DEAD Killed scheduled

5

5

5

5

5

RESPIRATION Sniffing

0

0

0

0

5

RESPIRATION Grunting

0

0

0

0

2

RESPIRATION Irregular

0

0

0

0

5

RESPIRATION Dyspnoea

0

0

0

3

5

RESPIRATION Shallow

0

0

0

1

0

RESPIRATION mouth breathing

0

0

0

0

4

MOUTH Salivation

0

0

0

0

3

BEHAVIOUR Hunched posture

0

0

0

3

5

BEHAVIOUR Hypoactive

0

0

0

0

5

BEHAVIOUR Ataxia

0

0

0

0

5

SKIN Piloerection

0

0

0

5

5

EYES Blepharospasm

0

0

0

1

5

Table 2: Clinical observations in females

Observation Type: All Types Sex: Female

From Day -2 (Start Date) to 7 (Start Date)

0 ppm

10

ppm

100

ppm

500

ppm

1000

ppm

DEAD Killed scheduled

5

5

5

5

5

RESPIRATION Sniffing

0

0

0

0

5

RESPIRATION Grunting

0

0

0

0

1

RESPIRATION Irregular

0

0

0

0

4

RESPIRATION Dyspnoea

0

0

0

0

5

RESPIRATION Shallow

0

0

0

0

1

RESPIRATION Sneezing

0

0

0

0

1

RESPIRATION mouth breathing

0

0

0

0

1

MOUTH Salivation

0

0

0

0

3

MOUTH chewing movement

0

0

0

0

2

BEHAVIOUR Hunched posture

0

0

0

0

5

BEHAVIOUR Hypoactive

0

0

0

0

5

BEHAVIOUR Ataxia

0

0

0

0

5

SKIN Piloerection

0

0

0

5

5

EYES Blepharospasm

0

0

0

0

5

Table 3: Body weight, males

Sex: Male

 

Bodywt

day-x(g)

 

[G]

Bodywt

day0(g)

 

[G]

Bodywt

 

(g)

 

[C]

Bodywt

 

(g)

 

[C]

-2

0

4

7

0 ppm

Mean

238.74

250.60

257.22

270.28

 

SD

12.47

12.92

16.19

16.66

 

N

5

5

5

5

10 ppm

Mean

241.14

251.68

255.16

266.16

 

SD

13.73

14.92

14.17

16.11

 

N

5

5

5

5

100 ppm

Mean

240.50

252.62

252.40

260.36

 

SD

16.63

17.44

15.54

16.57

 

N

5

5

5

5

500 ppm

Mean

239.66

248.42

237.30 **

237.44 *

 

SD

13.61

14.77

16.13

18.56

 

N

5

5

5

5

1000 ppm

Mean

241.08

250.74

218.26 **

210.72 **

 

SD

15.37

16.30

11.24

10.57

 

N

5

5

5

5

[G] - Ancova/Anova & Dunnett

[C] - Ancova/Anova & Dunnett {Covariate: Bodywt day 0}: * = p < 0.05; ** = p < 0.01

Table 4: Body weight, females

Sex: Female

 

Bodywt

day-x(g)

 

[G]

Bodywt

day0(g)

 

[G]

Bodywt

 

(g)

 

[C]

Bodywt

 

(g)

 

[C]

-2

0

4

7

0 ppm

Mean

165.04

164.42

168.86

179.60

 

SD

7.88

3.55

3.98

10.01

 

N

5

5

5

5

10 ppm

Mean

166.18

170.86

173.38

178.88

 

SD

8.08

5.82

4.21

6.97

 

N

5

5

5

5

100 ppm

Mean

165.56

167.18

171.60

176.16

 

SD

7.10

12.44

5.72

7.73

 

N

5

5

5

5

500 ppm

Mean

164.02

165.84

167.10

175.18

 

SD

9.77

9.40

9.52

9.71

 

N

5

5

5

5

1000 ppm

Mean

162.96

162.82

154.72 **

151.94 **

 

SD

8.33

8.84

8.40

9.01

 

N

5

5

5

5

[G] - Ancova/Anova & Dunnett

[C] - Ancova/Anova & Dunnett {Covariate: Bodywt day 0}: * = p < 0.05; ** = p < 0.01

Table 5: Body weight change, males

Sex: Male

 

 

Wgt change

last -x to0(g)

 

[G]

Body wt

change(g)

 

[G]

Body wt

change(g)

 

[G]

-2 - 0

0 - 4

4 - 7

0 ppm

Mean

11.86

6.62

13.06

 

SD

2.82

5.02

2.81

 

N

5

5

5

10 ppm

Mean

10.54

3.48

11.00

 

SD

3.05

3.27

3.24

 

N

5

5

5

100 ppm

Mean

12.12

-0.22

7.96

 

SD

1.60

3.58

2.57

 

N

5

5

5

500 ppm

Mean

8.76

-11.12 **

0.14 **

 

SD

1.66

3.07

3.54

 

N

5

5

5

1000 ppm

Mean

9.66

-32.48 **

-7.54 **

 

SD

2.05

5.39

3.31

 

N

5

5

5

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

Table 6: Body weight change, females

Sex: Female

 

 

Wgt change

last -x to0(g)

 

[G]

Body wt

change(g)

 

[G]

Body wt

change(g)

 

[G]

-2 - 0

0 - 4

4 - 7

0 ppm

Mean

-0.62

4.44

10.74

 

SD

6.62

3.58

6.49

 

N

5

5

5

10 ppm

Mean

4.68

2.52

5.50

 

SD

6.23

5.23

4.62

 

N

5

5

5

100 ppm

Mean

1.62

4.42

4.56

 

SD

8.31

9.42

2.77

 

N

5

5

5

500 ppm

Mean

1.82

1.26

8.08

 

SD

4.97

6.10

1.96

 

N

5

5

5

1000 ppm

Mean

-0.14

-8.10 *

-2.78 **

 

SD

9.85

3.41

2.18

 

N

5

5

5

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

Table 6: Food consumption, males

Sex: Male

Day(s) Relative to Animal Start Date

0 - 4

4 - 7

0 ppm

Mean

21.1

21.8

10 ppm

Mean

19.4

20.3

100 ppm

Mean

19.6

19.3

500 ppm

Mean

14.0

15.5

1000 ppm

Mean

7.5

9.0

Table 7: Food consumption, females

Sex: Female

Day(s) Relative to Animal Start Date

0 - 4

4 - 7

0 ppm

Mean

15.2

14.8

10 ppm

Mean

14.6

14.0

100 ppm

Mean

14.9

13.5

500 ppm

Mean

12.5

14.5

1000 ppm

Mean

6.2

7.8

Table 8: Relative organ weights, males

Sex: Male

 

 

 

 

 

 

 

 

Terminal bodywgt

(g)

 

[G]

Lungs rel.wgt

(g/kg bodywgt)

[G]

Heartrel.wgt

(g/kg bodywgt)

[G]

Adrenalsrel.wgt

(g/kg bodywgt)

[G]

Kidneysrel.wgt

(g/kg bodywgt)

[G]

Liverrel.wgt

(g/kg bodywgt)

[G]

Spleenrel.wgt

(g/kg bodywgt)

[G]

Testesrel.wgt

(g/kg bodywgt)

[G]

0 ppm

Mean

SD N

270.28

16.66

5

4.778

0.223

5

3.161

0.195

5

0.1974

0.0289

5

7.113

0.397

5

39.37

0.68

5

2.123

0.189

5

11.060

1.051

5

10 ppm

Mean SD

N

266.16

16.11

5

4.449

0.261

5

3.074

0.264

5

0.1929

0.0136

5

7.302

0.479

5

38.87

2.23

5

2.216

0.212

5

11.297

0.758

5

100 ppm

Mean SD

N

260.36

16.57

5

4.880

0.313

5

3.158

0.194

5

0.2370

0.0347

5

7.246

0.218

5

39.53

1.94

5

2.173

0.373

5

11.761

0.692

5

500 ppm

Mean

SD N

237.44 *

18.56

5

5.220

0.393

5

3.221

0.166

5

0.2560

0.0516

5

7.299

0.271

5

37.66

1.29

5

2.154

0.345

5

12.878 **

0.915

5

1000 ppm

Mean SD N

210.72 ** 10.57

5

6.235 ** 0.531

5

3.593*0.203 5

0.3132 **

0.0467

5

7.258

0.427

5

36.37*1.55 5

1.760

0.232

5

14.205 ** 0.444

5

[G] - Ancova/Anova & Dunnett: * = p < 0.05; ** = p < 0.01

Table 9: Relative organ weights, females

Sex: Female

 

 

 

 

 

 

 

 

Terminal bodywgt

(g)

 

[G]

Lungs rel.wgt

(g/kg bodywgt)

[G1]

Heartrel.wgt

(g/kg bodywgt)

[G]

Adrenalsrel.wgt

(g/kg bodywgt)

[G2]

Kidneysrel.wgt

(g/kg bodywgt)

[G]

Liverrel.wgt

(g/kg bodywgt)

[G]

Spleenrel.wgt

(g/kg bodywgt)

[G]

Ovaries rel.wgt

(g/kg bodywgt)

[G2]

0 ppm

Mean

SD N

179.60

10.01

5

5.518

0.189

5

3.434

0.116

5

0.3895

0.0695

5

6.974

0.342

5

37.49

0.89

5

2.256

0.243

5

0.4691

0.0414

5

10 ppm

Mean SD

N

178.88

6.97

5

5.230

0.256

5

3.561

0.186

5

0.4016

0.0434

5

7.402

0.517

5

39.61

1.90

5

2.135

0.193

5

0.4768

0.0334

5

100 ppm

Mean SD

N

176.16

7.73

5

5.534

0.231

5

3.420

0.179

5

0.4008

0.0399

5

7.477

0.447

5

39.29

2.02

5

2.093

0.168

5

0.5007

0.1116

5

500 ppm

Mean

SD N

175.18

9.71

5

5.687

0.227

5

3.378

0.100

5

0.4734

0.0867

5

7.148

0.294

5

40.03

3.53

5

2.209

0.109

5

0.4064

0.0352

5

1000 ppm

Mean SD N

151.94 ** 9.01

5

6.897 **

0.524

5

3.933 **

0.199

5

0.4276

0.1323

5

7.737

0.525

5

41.32

1.17

5

1.947

0.199

5

0.3969

0.0547

5

[G] - Ancova/Anova & Dunnett: ** = p < 0.01

[G1] - Ancova/Anova & Dunnett(Log): ** = p < 0.01

[G2] - Kruskal-Wallis & Dunnett on Ranks

Table 10: Macroscopic observations

Removal Reason(s): ALL

 

Number of Animals:

 

0 ppm

 

10

 

ppm

Male 100 ppm

 

500

 

ppm

 

1000

ppm

 

0 ppm

 

10

 

ppm

Female 100 ppm

 

500

 

ppm

 

1000

ppm

5

5

5

5

5

5

5

5

5

5

all organs/tissues

 

 

 

 

 

 

 

 

 

 

no visible lesions

5

5

4

4

-

5

4

5

3

2

lungs

 

 

 

 

 

 

 

 

 

 

incompletely collapsed

-

-

0

0

1

-

0

-

-

0

spots; red

-

-

1

1

4

-

1

-

-

1

lymph node, tracheobronchial

 

 

 

 

 

 

 

 

 

 

enlarged; unilateral

-

-

-

-

1

-

-

-

1

-

thymus

 

 

 

 

 

 

 

 

 

 

small

-

-

-

-

2

-

-

-

-

2

tissue not otherwise specified

 

 

 

 

 

 

 

 

 

 

neck; nodule; white, 2 mm

-

-

-

-

-

-

-

-

1

-

Table 11: Microscopic observations (excerpt)

Removal Reason(s): ALL

 

Number of Animals:

 

0 ppm

 

10

 

ppm

Male 100 ppm

 

500

 

ppm

 

1000

ppm

 

0 ppm

 

10

 

ppm

Female 100 ppm

 

500

 

ppm

 

1000

ppm

5

5

5

5

5

5

5

5

5

5

larynx

 

 

 

 

 

 

 

 

 

 

Examined

4

5

5

5

5

5

5

5

5

5

No Visible Lesions

2

1

4

1

0

4

5

5

1

0

inflammation; granulocytic

0

0

0

3

5**

0

0

0

1

3

.... minimal

0

0

0

1

0

0

0

0

1

1

.... mild

0

0

0

2

5

0

0

0

0

2

metaplasia; squamous

0

0

0

0

4*

0

0

0

0

4*

.... mild

0

0

0

0

4

0

0

0

0

4

ulceration

0

0

0

0

4*

0

0

0

0

0

.... minimal

0

0

0

0

1

0

0

0

0

0

.... mild

0

0

0

0

3

0

0

0

0

0

lungs

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

4

3

3

2

0

2

3

4

4

0

haemorrhage(s); alveolar

0

0

0

0

4*

2

0

0

0

0

bronchus; inflammation; granulocytic

0

0

0

1

4*

0

0

0

1

4*

.... minimal

0

0

0

1

4

0

0

0

1

3

 

nose, level 1

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

5

4

5

3

1

5

3

4

2

3

inflammation; granulocytic

0

1

0

2

4*

0

2

0

3

2

.... minimal

0

1

0

1

2

0

2

0

2

2

.... mild

0

0

0

1

2

0

0

0

1

0

nose, level 2

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

5

5

5

1

0

5

5

5

0

0

inflammation; granulocytic

0

0

0

3

5**

0

0

0

5**

5**

.... minimal

0

0

0

3

0

0

0

0

5

0

.... mild

0

0

0

0

0

0

0

0

0

1

.... moderate

0

0

0

0

2

0

0

0

0

4

.... marked

0

0

0

0

3

0

0

0

0

0

ulceration

0

0

0

0

5**

0

0

0

0

5**

.... mild

0

0

0

0

0

0

0

0

0

2

.... moderate

0

0

0

0

1

0

0

0

0

3

.... marked

0

0

0

0

4

0

0

0

0

0

necrosis

0

0

0

0

5**

0

0

0

0

5**

.... mild

0

0

0

0

0

0

0

0

0

1

.... moderate

0

0

0

0

3

0

0

0

0

1

.... marked

0

0

0

0

2

0

0

0

0

3

metaplasia; squamous

0

0

0

0

3

0

0

0

2

5**

.... minimal

0

0

0

0

0

0

0

0

2

0

.... mild

0

0

0

0

1

0

0

0

0

3

.... moderate

0

0

0

0

2

0

0

0

0

2

nose, level 3

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

5

5

1

1

0

5

5

0

0

0

inflammation; granulocytic

0

0

0

2

5**

0

0

0

1

5**

.... minimal

0

0

0

2

0

0

0

0

1

0

.... mild

0

0

0

0

3

0

0

0

0

4

.... moderate

0

0

0

0

2

0

0

0

0

1

ulceration

0

0

0

0

5**

0

0

0

0

2

.... mild

0

0

0

0

2

0

0

0

0

2

.... moderate

0

0

0

0

3

0

0

0

0

0

necrosis

0

0

0

0

1

0

0

0

0

1

.... mild

0

0

0

0

1

0

0

0

0

1

nose, level 3 (Continued...)

 

 

 

 

 

 

 

 

 

 

olfactory epithelium; atrophy

0

0

4*

3

4*

0

0

5**

5**

4*

.... minimal

0

0

4

1

0

0

0

5

5

2

.... mild

0

0

0

2

2

0

0

0

0

2

.... moderate

0

0

0

0

2

0

0

0

0

0

metaplasia; squamous

0

0

0

1

4*

0

0

0

0

5**

.... mild

0

0

0

1

2

0

0

0

0

1

.... moderate

0

0

0

0

2

0

0

0

0

4

nose, level 4

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

4

5

2

0

0

5

5

0

0

0

inflammation; granulocytic

0

0

0

2

5**

0

0

0

2

5**

.... minimal

0

0

0

2

0

0

0

0

2

0

.... mild

0

0

0

0

4

0

0

0

0

5

.... moderate

0

0

0

0

1

0

0

0

0

0

ulceration

0

0

0

0

5**

0

0

0

0

4*

.... minimal

0

0

0

0

0

0

0

0

0

1

.... mild

0

0

0

0

3

0

0

0

0

3

nose, level 4 (Continued...)

 

 

 

 

 

 

 

 

 

 

.... moderate

0

0

0

0

1

0

0

0

0

0

.... marked

0

0

0

0

1

0

0

0

0

0

olfactory epithelium; atrophy

0

0

3

4*

4*

0

0

4*

5**

3

.... minimal

0

0

2

2

3

0

0

3

1

2

.... mild

0

0

1

2

1

0

0

1

4

1

metaplasia; squamous

0

0

0

0

5**

0

0

0

0

5**

.... minimal

0

0

0

0

1

0

0

0

0

0

.... mild

0

0

0

0

4

0

0

0

0

4

.... moderate

0

0

0

0

0

0

0

0

0

1

nose, level 5

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

5

5

4

3

0

5

5

5

3

0

inflammation; granulocytic

0

0

0

0

5**

0

0

0

0

4*

.... minimal

0

0

0

0

1

0

0

0

0

2

.... mild

0

0

0

0

4

0

0

0

0

2

ulceration

0

0

0

0

2

0

0

0

0

2

.... minimal

0

0

0

0

1

0

0

0

0

0

.... mild

0

0

0

0

0

0

0

0

0

2

.... moderate

0

0

0

0

1

0

0

0

0

0

nose, level 5 (Continued...)

 

 

 

 

 

 

 

 

 

 

metaplasia; squamous

0

0

0

0

5**

0

0

0

0

5**

.... minimal

0

0

0

0

1

0

0

0

0

0

.... mild

0

0

0

0

4

0

0

0

0

5

nose, level 6

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

5

5

5

4

0

5

5

5

5

3

trachea/bronchi

 

 

 

 

 

 

 

 

 

 

Examined

5

5

5

5

5

5

5

5

5

5

No Visible Lesions

4

4

5

1

0

5

5

5

1

1

inflammation; granulocytic

0

0

0

3

5**

0

0

0

0

4*

.... minimal

0

0

0

1

0

0

0

0

0

1

.... mild

0

0

0

2

5

0

0

0

0

3

inflammation; mixed

0

0

0

1

0

0

0

0

2

0

.... minimal

0

0

0

1

0

0

0

0

1

0

Fisher's Exact: * = p < 0.05; ** = p < 0.01

Conclusions:
Under the conditions of this 7-day concentration range-finding study, inhalation exposure to the test substance at target concentrations of 10, 100, 500 and 1000 ppm resulted in the following treatment-related changes:
• At 1000 ppm, marked toxicity was observed; in nearly all animals, dyspnea, sniffing, irregular respiration, hunched posture, ataxia, blepharospasm, piloerection and hypoactivity were observed. Body weight of males exposed to the top concentration were 22% lower than controls (on day 7); for females the reduction in body weight was 15%. Relative weight of the heart and lungs was increased in male and female animals. When assessing histopathology, severe olfactory epithelial atrophy was observed in the nasal tissues. Milder effects were observed in the larynx, trachea/bronchi and lungs, mainly of inflammatory nature.
• At 500 ppm, transient clinical abnormalities (dyspnea, shallow breathing, hunched posture and piloerection) were observed in males especially. Minimal to mild atrophy of the nasal olfactory epithelium was observed in most animals when assessing nose tissue levels 3 and 4 (out of the 6 levels assessed).
• At 100 ppm, minimal atrophy of the nasal olfactory epithelium was still observed in most animals, with 2 cases of mild atrophy observed at a single level of the nose in 2 animals.
• At 10 ppm, no exposure-related toxicity was seen. Thus, the No-Observed-Adverse-Effect- Concentration (NOAEC) in this range finding study was placed at 10 ppm (target concentration).

Based on these observation, the target concentration for the main study were selected at 10, 50, and 250 ppm. Sub-acute exposure to the test substance at 250 ppm is anticipated to result in clear respiratory toxicity (without causing lethality or severe distress), while at 10 ppm exposure-related adverse changes are no longer expected.
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Name1 : Dimethylethylamine
Chemical name 1 : Ethyldimethylamine; N,N-Dimethylethylamine
CAS registry number1 : 598-56-1
Molecular formula1 : C4H11N
Structural formula1 :
Molecular weight1 : 73.13 g/mol
Batch/lot number1 : SAMP160321
Purity1 : 99.41%
Solubility in water1 : completely soluble; exothermic reaction!
Melting point1 : -140 °C
Boiling point1 : 36.3 °C
Vapor pressure 1 : 527 hPa at 20° C
Density 1 : 660 kg/m3 at 20 °C
Viscosity1 : 0.67 mPa.s (OECD Test Guideline 114)
Flash Point1 : -25°C closed cup (ISO 3679)
Quantity received : 6.75 kg
Supplier : Arkema
Approved for use until1 : 20 November 2018
Storage conditions : ambient temperature (15-25°C)
Date of receipt : 23 November 2017
1Characteristics provided by the sponsor
The Triskelion dispense reference number is 17025D.
Species:
rat
Strain:
Wistar
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 regulatory authorities. Young adult, male and female Wistar outbred (Crl:WI(Han)) rats were obtained from a colony maintained under specific pathogen free (SPF) conditions by Charles River Laboratories. The Wistar rat strain was used because it is routinely used at the test facility for this type of study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
The age of the rats was about 7-8 weeks on the day of randomization (8 January and 29 March 2018 for animals of the range finding and main study, respectively). Body weight at allocation was within ±20% of the mean weight for each sex. Mean body weights at the start of exposure (day 0) in the range finding study were 251 and 166 grams for male and female animals, respectively. Mean body weights at the start of exposure in the main study were 235 and 170 grams for male and female animals, respectively.

Upon arrival on 21 March 2018, the rats (33 males and 33 females) were taken in their unopened shipping containers to a quarantine room (animal room 5.1.14) and were checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from five randomly selected animals. On 23 March 2018, the results of the serological examinations were received and indicated an acceptable microbiological status. The animals were subsequently released for experimental use, and were transferred to their definitive room (animal room 6.0.06). The duration of the acclimatization period to the laboratory conditions prior exposure (period between arrival and the start of the exposure period) was 13 days.

Shortly before initiation of exposure (on 29 March 2018), the animals were allocated to the various groups by computer randomization proportionally to body weight (males and females separately). The surplus animals (3 males, 3 females) were kept in reserve to serve as sentinels. These animals were not used in the present study.

Animal husbandry
Animal room
From their arrival, the animals were housed under conventional conditions in one animal room separated by sex. No other test system was housed in the same room during the study. The room was ventilated with about 10 air changes per hour and was maintained at a temperature of 20-24oC and a relative humidity of 45-65%. The upper limit of relative humidity was, however, higher than 65% for short periods of time because of wet cleaning activities. Relative humidity was below 45% on 31 March and 3 April 2018 (briefly, with a minimum of 44%) and on multiple occasions for prolonged durations in the period 5 April – 24 May 2018 (minimum of 39%). Lighting was artificial with a sequence of 12 hours light and 12 hours dark.

Caging
During exposure, the animals were housed individually in the inhalation unit. Immediately after each exposure, the animals were returned to their home cages. When not exposed, the animals were housed five animals to a cage, separated by sex. All animals were housed in macrolon cages with wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany) as bedding material and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. The cages and bedding were changed at least weekly.

Food and drinking water
Food was provided ad libitum from the arrival of the animals until the end of the study, except during exposure and – for animals of the main study – the overnight fasting period before sacrifice. The animals received a cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDS Special Diets Services, Whitham, England). Each batch of VRF1 (FG)) diet is analyzed by the supplier for nutrients and contaminants. The food was provided as a powder in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. The food in the feeders was replaced with fresh portions once weekly and filled up as needed.

Drinking water was provided ad libitum from the arrival of the animals until the end of the study, except during exposure. Each cage was supplied with 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. Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the test facility. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of the
test facility for a limited number of variables.

Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Exposure equipment
The animals were exposed to the test atmosphere in nose-only exposure units, in an illuminated laboratory room different from the room where the animals were housed. Animals of groups 2, 3, 4 and – for the range finding – 5 were exposed in inhalation chambers consisting of a cylindrical aluminium column, surrounded by a transparent cylinder (a modification of the chamber made by ADG Developments Ltd., Codicote, Hitchin, Her ts, SG4 8UB, United
Kingdom). The column had a volume of 46.7 L and consisted of a top assembly with the entrance of the unit, a mixing section, two rodent tube sections and at the bottom the base assembly with the exhaust port. Each rodent tube section had 20 ports for animal exposure. Control animals (group 1) were exposed to clean air in a polypropylene nose-only inhalation chamber with a volume of 48.2 L (manufactured by P. Groenendijk Kunststoffen BV) which was very similar in construction to the aluminium chambers described above.

Empty ports were used for test atmosphere sampling (for analysis of the actual concentration) and measurement of oxygen, carbon dioxide, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. Animals were rotated with respect to their position in the column every week (main study) or two days (range finding study). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. Habituation to the restraint in the animal holders was not performed because in our experience habituation does not help to reduce possible stress (Staal et al., 2012).

In our experience, the animal’s body does not exactly fit in the animal holder which always results in some leakage from the high to the low pressure side. By securing a positive pressure in the central column and a slightly negative pressure in the outer cylinder, which encloses the entire animal holder, dilution of test atmosphere by air leaking from the animals’ thorax to the nose was avoided. The unit was illuminated externally by normal laboratory fluorescent tube lighting. The total air flow through the unit was at least 1 liter/min for each rat. The air temperature and relative humidity in the unit were maintained at 22 ± 3°C and between 30 and 70%, as far as possible.

Generation of the test atmosphere
The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. A schematic diagram of the generation and the exposure system is presented in Figure 1. To generate the test atmospheres, a continuous flow of liquid test material3, controlled by a peristaltic pump (Minipulse 3, Gilson, Velliers le Bel, France) was allowed to evaporate in a mass flow controlled (Bronkhorst Hi Tec, Ruurlo, the Netherlands) stream of compressed dry air, by directing it through a glass evaporator which was kept at a constant temperature of 48°C by circulating heated water. The resulting single stream of concentrated vapor was led through a condense trap (not used during the range finding study) and was subsequently divided for the different groups using mass flow controllers (Bronkhorst Hi Tec) and mixed with a controlled stream of humidified compressed air via an eductor (Fox Valve Development Corp., Dover, NJ, USA). The eductors were calibrated by measuring the total air flow at a range of driving air pressures of the eductors encompassing the driving pressures used during the study; the driving air pressure was used to monitor the total flow. The resulting test atmosphere was led to the inlet at the top of the exposure chamber and directed downward towards the noses of the animals; the atmosphere was exhausted at the bottom. As of 9 April 2018, a PI feedback system was installed to automatically control the feedrate of the peristaltic pump, based on the measured actual concentration (see paragraph 4.10.1). The feedback system took into account the proportional (P) and the integrated deviations (I) of the concentrations from the setpoint.

The exposure chamber for the control animals (group 1) was supplied with a stream of humidified compressed air only, which was controlled by a reducing valve and measured by 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of exposure conditions
Actual concentration
The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (groups 2 and 3 of the main study and group 3 of the range finding study: Ratfisch RS55T, Munich, Germany; remaining groups: 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 Data acquisition system (range finding study: CAN transmitters, G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany; main study: MyriaNed, van Mierlo Ingenieursbureau BV, Eindhoven, the Netherlands). 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.

Prior to the first exposure, the output of the flame ionization detector of the TCA was calibrated using gas sample bags. To this end, sample bags were filled with an accurate (mass flow controlled) volume of air and an accurate (weighed) amount of test material, injected into the bag through a septum. Three concentrations were thus prepared (at least in duplicate) – at approximately 80%, 100% and 120% of the target concentration of each group – and analyzed by the TCA. Linear relations were found between the response of the analyzers and the concentration of the test material.

The calibrations were checked weekly during the study. To this end, gas sample bags were prepared at each target concentration as described above, and were subsequently analyzed by the TCA. If the measured concentration deviated more than 5% from the calculated concentration, the calibration check was repeated. If the deviation was more than 5% at the re-check, a complete re-calibration was to be carried out (which was not necessary during the range finding nor the main study).
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
5 days per week, over a 28-day study period (20 exposure days in total)
Dose / conc.:
0 ppm (analytical)
Dose / conc.:
10 ppm (analytical)
Remarks:
30 mg/m3
Dose / conc.:
49.7 ppm (analytical)
Remarks:
149.1 mg/m3
Dose / conc.:
249 ppm (analytical)
Remarks:
747 mg/m3
No. of animals per sex per dose:
The 28-day study comprised four test groups of five male and five female rats each, i.e. a control group exposed to clean air and three groups exposed to different concentrations of the test material.

Five additional males and 5 additional females were included in the control and high-concentration group, which were exposed similarly and kept for a recovery period of 28 days after the last exposure.
Control animals:
yes, concurrent no treatment
Details on study design:
7-day concentration range finding study:
The range finding study consisted of five test groups of five male and five female rats each, i.e. a control group exposed to clean air, and four groups exposed to different concentrations of the test material. The highest concentration was intended to result in clear toxicity but not death or moribundity. The lowest concentration was intended to produce little or no evidence of toxicity. The animals of the control group were handled identically as those of the other groups, except for exposure to the test material. The animals were exposed for 6 hours per day, 5 days per week, over a 7-day study period (5 exposure days in total) and sacrificed on the day after the last exposure. The date of the first exposure (10 January 2018) was named day 0.

Concentrations (ppm): 0, 10, 100, 500 and 1000

Selection of target concentrations for the concentration range finding study were based on the 4h LC50 that was found to be ca. 3400 ppm; no mortality was observed at a lower concentration of 2300 ppm. Although no repeated-dose inhalation toxicity data were available for Dimethylethylamine, there were data for the toxicity of the comparable substance Trimethylamine (CAS no. 75-50-3). The 4h LC50 was comparable at ca. 2400-3500 ppm. In a subacute study, mild toxicity was observed at the low concentration of 75 ppm, while mild toxicity was observed at a concentration as low as 10 ppm in a chronic study (all information available at http://www.echa.eu); thus, to enable identification of a NOAEC and characterize the concentration-response relationship, a broad concentration range was chosen for this range finding study.


Animal allocation
7-day range finding study
27 male and 27 female rats were ordered, and upon arrival on 20 December 2017 they were taken in their unopened shipping containers to a quarantine room (animal room 5.1.20) and were checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from four randomly selected animals. On 22 December 2017, the results of the serological examinations were received and indicated an acceptable microbiological status. The animals were subsequently released for experimental use, and they were transferred to their definitive room (animal room 6.0.06) on the same day. The duration of the acclimatization period to the laboratory conditions prior exposure (period between arrival and the start of the exposure period) was 21 days.

On 8 January 2018, shortly before initiation of exposure of the rats, they were allocated to the various groups by computer randomization proportionally to body weight. . The surplus animals (2 males, 2 females) were kept in reserve to serve as sentinels. These animals were not used in the present study.

28-day main study
Upon arrival on 21 March 2018, the rats (33 males and 33 females) were taken in their unopened shipping containers to a quarantine room (animal room 5.1.14) and were checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status was conducted in blood samples taken from five randomly selected animals. On 23 March 2018, the results of the serological examinations were received and indicated an acceptable microbiological status. The animals were subsequently released for experimental use, and were transferred to their definitive room (animal room 6.0.06). The duration of the acclimatization period to the laboratory conditions prior exposure (period between arrival and the start of the exposure period) was 13 days.

Shortly before initiation of exposure (on 29 March 2018), the animals were allocated to the various groups by computer randomization proportionally to body weight (males and females separately). The surplus animals (3 males, 3 females) were kept in reserve to serve as sentinels. These animals were not used in the present study.

4.6 Identification
The study was identified as Triskelion study number 21124, with a subcode /01 for the range finding study and /02 for the main study.

Before and during allocation, the individual rats were identified by a transient mark on their tail.

After allocation, they were identified by an identification number (even for males and odd for females), using subcutaneous transponders. During the study each group of rats was coded by a number and a color. Each cage was provided with a card showing the color code, the animal identification numbers, the group number and the study number.
Positive control:
None
Observations and examinations performed and frequency:
Clinical observations
Animals were observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. The observations included – but were not limited to – the signs listed in Annex 6. 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 hindered 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 examination (28-day sub-acute study)
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 groups (on day 23). Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate. Since no evident exposure-related ocular changes were observed, eye examinations were not extended to the animals of the intermediate concentration groups at the end of the exposure period, or to animals of the recovery groups.

Body weights
In the range finding study, the body weight of each animal was recorded 2 days before the start of the exposure. These pre-test weights served as a basis for animal allocation. Subsequently, the animals were weighed prior to the first exposure on day 0, on day 4 of exposure and on their scheduled sacrifice data in order to calculate the correct organ to body weight ratios.

In the main study, the body weight of each animal was recorded 5 days before the start of exposure. These pre-test weights served as a basis for animal allocation. Subsequently, the animals of the main study were weighed prior to exposure on the first day (day 0), and twice a week thereafter. 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.

Food consumption
Food consumption of the animals was measured per cage by weighing the feeders. The results are expressed in g per animal per day. Food consumption was measured twice weekly for animals of the range finding study. For animals of the main study, food consumption was measured once weekly.

Estrus cycle evaluation (28-day sub-acute study)
Vaginal smears to evaluate the estrus cycle length and normality were made daily in the three weeks prior to sacrifice, including the day of sacrifice, in all female animals of the main study. Since no exposure-related abnormalities were observed in animals of the main study, estrus cycle evaluation was not extended to animals of the recovery groups.
Sacrifice and pathology:
Sperm analysis (28-day sub-acute study)
Epididymal sperm motility, count and morphology At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all male animals from both the main and recovery groups (30 animals in total). For this purpose the cauda epididymis was dissected, weighed and thereafter minced in M199 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) were measured for all males, using the Hamilton Thorne Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained, and two hundred spermatozoa of this smear were examined for morphology.

Testicular sperm count
At scheduled necropsy, the left testis of all males were placed on dry ice and subsequently stored in a freezer (<-70°C) for later determination of the number of homogenization-resistant spermatids. The testes to be analysed were thawn just before further processing. Following removal of the tunica albuginea, the testicular parenchyma were weighed, minced and homogenized in Saline Triton X-100 solution. Following DNA-staining, the homogenizationresistant sperm heads were enumerated using the IVOS. The daily sperm production was calculated. The evaluation of homogenization-resistant spermatids was performed in the males of the control and high-concentration groups from both the main and recovery study. Since no exposure-related changes were observed in animals of the high-concentration group, the evaluation of homogenization-resistant spermatids was not extended to animals of the intermediate-concentration groups.

Hematology (28-day sub-acute study)
Hematology was conducted at the end of the treatment period on all surviving animals of the main groups. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anesthesia at sacrifice. Citrate (for prothrombin time) or EDTA (for other parameters) were used as anticoagulant. Blood samples were discarded after analysis. In each sample the following determinations were carried out.
- hemoglobin
- packed cell volume
- red blood cell count
- reticulocytes
- total white blood cell count
- differential white blood cell count
(lymphocytes, neutrophils, eosinophils, basophils and monocytes)
- prothrombin time
- thrombocyte count (platelet count)

The following parameters were calculated:
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin (MCH)
- mean corpuscular hemoglobin concentration (MCHC)

Since (possible) exposure-related changes were observed in animals of the main groups, investigation of all hematology parameters (except prothrombin time) was extended to animals of the recovery groups.

4.11.8 Clinical chemistry (28-day sub-acute study)
Clinical chemistry was conducted at the end of the exposure period on all surviving rats after overnight fasting, at the same time blood samples for hematology were collected. Blood samples were taken from the abdominal aorta of the rats whilst under pentobarbital anaesthesia. The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. Plasma samples were stored frozen (<-18°C) until analysis and
discarded afterwards.
- alkaline phosphatase activity (ALP) - bilirubin (total)
- aspartate aminotransferase activity (ASAT) - cholesterol
- alanine aminotransferase activity (ALAT) - triglycerides
- gamma glutamyl transferase activity (GGT) - calcium (Ca)
- total protein - sodium (Na)
- albumin - potassium (K)
- ratio albumin to globulin (calculated) - chloride (Cl)
- urea - inorganic phosphate (PO4)
- creatinine - thyroxine (T4)
- glucose (fasting)

Since (possible) exposure-related changes were observed in animals of the main groups, investigation of clinical chemistry parameters was extended to animals of the recovery groups.

Pathology
Sacrifice, organ weights and macroscopic examination
At the end of the exposure period, the animals were sacrificed 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 28-day post-exposure recovery period included in the sub-acute study. The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes.

7-day concentration range finding study
The following organs of all 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 weights and the terminal body weight:
- heart
- adrenals
- kidneys
- liver
- spleen
- testes
- ovaries
- lungs with trachea

28-day sub-acute study
The following organs of all 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 weights and the terminal body weight:
- adrenals
- brain
- heart
- kidneys
- liver
- lungs (left lung only, because of lavage of the other lobes)
- spleen
- testes
- ovaries
- thymus

For assessment of fertility in male rats of the main groups (20 animals in total), the following additional weights were determined:
- epididymides
- prostate
- seminal vesicles

Since (possible) exposure-related changes were observed in animals of the main groups, the seminal vesicles (+ coagulation glands), testes, epididymides (all three organs for males only) and thymus (both sexes) were also weighed in animals from the recovery groups of the main study; the other organs included for main study animals were not weighed.

Bronchoalveolar lavage and measurements (28-day sub-acute study)
The lungs of all animals of the main groups were lavaged at necropsy according to a standardized method. In short: the right half of the lungs (after binding off the left lung lobe, used for histopathology) from these animals was rinsed three times with a single volume of 26.7 ml saline per kg body weight (one value for each group based on mean body weight). The final amount of lung lining fluid and cells collected was weighed and retained on ice. The
bronchoalveolar lavage cells were recovered by centrifugation (250xG) for 5 minutes. The temperature control of the centrifuge was set at 4ºC. Each cell pellet thus obtained per animal was resuspended in 0.5 ml saline and used for total white blood cell numbers, viability and cell differentials. The supernatant was stored frozen (<-18°C) until analysis of biochemical parameters. BAL fluid samples were discarded after analysis.

Cellular determinations
Total white blood cell numbers were counted using an AdVia 2120i analyzer (Siemens N.V., the Netherlands; Reference: “Training manual 04/11/99, chapter 5 Impedance”.) The number of viable cells was determined using an acridine orange / ethidium bromide staining method in combination with fluorescent microscopic evaluation. The cytospins for cell differentials were made using a Cyto-Tek (Sakura, Netherlands) and stained by May-Grunwald Giemsa. The differential cells (monocytes, macrophages, neutrophils, eosinophils, lymphocytes) were evaluated by light microscopy (absolute numbers were calculated from total white blood cell number and percentage distribution of the different cell types).

Since exposure-related changes were observed in animals of the main groups, investigation of bronchoalveolar lavage parameters was extended to animals of the recovery groups of the sub-acute study.


Histopathology
7-day concentration range finding study
For histopathological examination, samples of the following tissues and organs from all animals, and all gross lesions were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of Formalin). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to ensure fixation). Tissues were processed and examined using the same methods as described below for the relevant tissues examined during the 28-day study. The carcass containing any remaining tissues was retained in the fixative until completion of the histopathological examination and then discarded.
- kidneys
- nasal turbinates
- larynx
- trachea
- lungs
- tracheobronchial lymph nodes

All preserved tissues of all animals of the control and top-concentration groups were examined histopathologically (by light microscopy). Since exposure-related changes were observed in the nasal tissues (all 6 levels), larynx, trachea and lungs of the top-concentration group, histopathological examination of these tissues was extended to animals of the intermediate concentration groups of the range finding study.

28-day sub-acute study
For histopathological examination, samples of the following tissues and organs from all animals, and all gross lesions were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of Formalin). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to insure fixation).
- adrenals
- bone marrow
- brain (including sections of cerebrum, cerebellum, and medulla/pons)
- eyes (with specific focus on adverse corneal changes)
- heart
- kidneys
- liver
- esophagus
- ovaries
- seminal vesicles
- spinal cord (cervical, mid-thoracic, and lumbar)
- spleen
- stomach
- testes (including testicular staging)
- thymus
- thyroid
- tracheobronchial lymph nodes
- uterus
- respiratory tract including larynx, trachea, left lung lobe (the right lobes were lavaged) and
nasopharyngeal tissues
- all gross lesions

The carcass containing any remaining tissues was also retained in Formalin, but discarded after completion of the histopathological examination.

Slide preparation
Tissues to be examined were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. Unless required for histopathological examination, the tissues of the animals of the intermediate concentration groups (groups 2 and 3) were not processed, except for the noses of animals of groups 2 and 3 which were decalcified and embedded in paraffin concurrently with the noses of the animals of groups 1 (control) and 4 (high concentration).

Histopathological examination
All preserved tissues of all animals of the control and high-concentration main groups were examined histopathologically (by light microscopy). In addition, all gross lesions observed in rats of the intermediate-concentration main groups were examined microscopically. Histopathology was subjected to a peer review system.

The nasopharyngeal tissues were examined at six levels (Woutersen et al., 1994; an illustration of these levels is shown in Annex 9) with one level to include the nasopharyngeal duct and the Nasal Associated Lymphoid Tissue (NALT), the larynx at three levels (one level to include the base of the epiglottis). The trachea was examined at three levels (including the bifurcation, and one longitudinal section through the carina), and the left lung lobe at three levels. When examining the eye, particular focus was directed towards assessment of corneal changes, which have been observed in exposed workers who have experienced “blue haze”, associated with
corneal swelling.

Since exposure-related changes were observed in the nasal tissues of animals of the highconcentration group, histopathological examination of these tissues was extended to animals of the intermediate concentration main groups (nose levels 2-6) and to animals of the recovery groups (nose levels 1-6) of the 28-day sub-acute study.
Statistics:
Ancova/Anova & Dunnett
Pretreatment body weight, body weight after initiation of treatment, clinical pathology (hematology, clinical chemistry), bronchoalveolar lavage parameters, organ weights, sperm motility
(numerical), sperm count, testicular sperm count.
Food consumption - no statistics were applied (only one cage per sex per group).
Kruskal-Wallis & Wilcoxon: Estrus cyclicity: mean length of the longest cycle, number of complete cycles in the test period; sperm motility (expressed as %), sperm morphology
Chie-squared & Fisher's Exact: Incidences of histopathological changes; Estrus cyclicity: number of acyclic females, number of animals with prolonged estrus period
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No exposure-related clinical abnormalities were observed. The few signs noted were considered unrelated to the exposure to the test material. Abnormalities of the skin or fur (sparsely haired areas, encrustations) were observed in a few animals across the groups. These are common findings, possibly caused by slight movement of the animals in the restraining tubes during exposure, resulting in slight irritation of the skin.
Mortality:
no mortality observed
Description (incidence):
All animals survived until scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Male animals of the high-concentration group showed a statistically significantly lower body weight than controls from day 3 until the end of the exposure period (up to ~10% lower average body weight by day 24), which was the result from body weight loss or decreased growth on days of exposure; normal growth was observed during periods including the exposure-free weekends. The difference in body weight compared to control animals gradually decreased during the recovery period. A trend towards decreased body weight gain was also observed in females of the high-concentration group and in males of the mid-concentration group during the first ~2 weeks of exposure, but statistical significance was not reached.

PLEASE REFER TO ATTACHED TABLES - 'Body weight' and Body weight change'
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Although statistical analysis could not be performed (as only one cage per sex per group was used), food consumption of male animals of the high-concentration group was slightly lower (maximally approximately 15%) than controls throughout the exposure period; normal food intake was observed during the recovery period. Food consumption in females of the high-concentration group also seemed slightly decreased (maximally approximately 10%) during the first half of the exposure period, when compared to controls. No treatment-related changes in food intake were observed in animals of the low- and midconcentration group.

PLEASE REFER TO ATTACHED TABLE - 'Food consumption'
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Ophthalmoscopic examination near the end of the exposure period revealed unilateral focal corneal opacity in two (of five) male animals of the high-concentration main group. This – usually transient, reversible – lesion is occasionally observed as a background finding, possibly related to the nose-only mode of exposure in restraining tubes (sometimes causing minor superficial damage to the cornea, because the animal’s head may be somewhat compressed against the front of the tube). For this reason, and because the lesion was observed only focally (rather than diffusely throughout the cornea), in just two animals of the high-concentration group, and was not associated with any microscopic changes in the eye, a relation with the exposure to the test material was considered to be highly unlikely. No further ophthalmoscopic abnormalities were observed at the end of the exposure period.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Analysis of hematology parameters revealed the following statistically significant differences between animals of the main groups exposed to the test material and unexposed controls:
- Decreased absolute number of eosinophils in males of the high-concentration group. This finding was not accompanied by any changes in other white blood cell parameters, or by a shift in the percentage distribution of differential white blood cell numbers and might be a consequence of stress.
- Increased packed cell volume (PCV) in males of the low-concentration group, which was considered to be a chance finding (unrelated to the exposure), because a concentration-response relationship was absent.
There were no statistically significant changes in coagulation parameters of animals of the main groups.

Investigation of hematology parameters in animals of the recovery groups, performed in response to the statistically significant differences observed in animals of the main groups, did not reveal any exposure-related changes. In the absence of any corresponding changes at the end of the exposure period or any corroborative findings in animals of the recovery groups, a slightly increased mean corpuscular volume (MCV) in males of the high-concentration (without any changes in parameters from which MCV is calculated, i.e. PCV and total red blood cell numbers) and slightly lower lymphocyte and total white blood cell numbers in females of the
high-concentration recovery group were considered to be chance findings, unrelated to the exposure to the test material.

PLEASE REFER TO ATTACHED TABLES - 'Red blood cell and coagulation parameters (main and recovery)', 'Total and differential white blood cell counts (main and recovery)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Analysis of clinical chemistry parameters revealed the following statistically significant differences between animals of the main groups exposed to the test material and unexposed controls:
- Decreased plasma activity of alkaline phosphatase (ALP) in males of all dose groups. This difference was considered to be a chance finding (unrelated to the exposure), caused by a few relatively high values recorded for concurrent control animals, which was substantiated by the results obtained in recovery animals (showing a control range of plasma ALP activity similar to exposed animals of the main groups). Moreover, an increase – rather than a decrease – in ALP activity is commonly associated with pathological changes. Thus, the apparent decrease in ALP activity in exposed males of the main groups was considered to be of no toxicological relevance.

- Increased thyroxine (T4) concentration in plasma of females of the high-concentration group. Though increased relative to concurrent controls, the average T4 concentration (466.22 ± 133.43 ng/mL) was within the range of recent historical controls and in line with results obtained in animals of the recovery group.

- Increased plasma sodium (Na) concentration in females of the mid-concentration group, which was considered to be a chance finding, because a concentration-response relationship was absent.

Since statistically significant differences were observed in animals of the main groups, clinical chemistry parameters were also examined in animals of the recovery groups. No exposure-related changes were observed at the end of the recovery period. A few statistically significant differences in females of the high-concentration group when compared to concurrent controls (slightly lower ALP activity, slightly lower average plasma albumin concentration and – consequently – a decreased albumin/globulin ratio) were considered chance findings, since corresponding changes were not observed at the end of the exposure phase.

PLEASE REFER TO ATTACHED TABLES - 'Clinical chemistry (main and recovery)'
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):
Organ weight data obtained at necropsy of animals of the main groups at the end of the exposure period showed a statistically significant increase in relative weight of the testes and adrenals in males of the high-concentration group. Since the absolute weight was unchanged, this change is considered secondary to the reduced body weight. Stress might have enhanced the effect on adrenal weights. There was a decrease in absolute and relative weight of the thymus in females of the high-concentration group. Thymus weight also seemed decreased in males of the high-concentration group, but statistical significance was not reached. No exposure-related changes were observed in the weight of selected organs (seminal vesicles, testes, epididymides, and thymus) of animals sacrificed at the end of the recovery period.
In the absence of any changes after the recovery phase and no histopathological findings, a lower thymus weight was likely stress related caused by inhalation of a caustic substance, and was considered treatment-related but non-adverse.

A few statistically significant changes in organ weight of females of the low-concentration group (decreased absolute spleen weight, decreased absolute and relative thymus weight) were considered chance findings, because a concentration-response relationship was absent.

PLEASE REFER TO ATTACHED TABLES - 'Absolute organ weights (main and recovery)' 'Relative organ weights (main and recovery)'
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy, no exposure-related macroscopic changes were observed in the animals of the main and recovery groups. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in the different groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic evaluation revealed an exposure-related, concentration-dependent increase of degeneration of the respiratory and olfactory epithelium in the nasal tissues of animals of the mid- and high-concentration groups sacrificed at the end of the exposure period. In the highconcentration group, all animals showed minimal to mild degeneration of the respiratory epithelium in the rostral parts of the nose (level 2) and mild to moderate degeneration of the olfactory epithelium in the dorsocaudal parts of the nose (levels 3-6). The incidence and severity of these lesions was lower in animals of the mid-concentration group, with minimal to mild degeneration of the respiratory epithelium observed at level 2 in 4/10 animals, and minimal to mild degeneration of the olfactory epithelium observed at levels 3 (10/10 animals), 4 (9/10 animals) and 5 (2/10 animals). No exposure-related nasal pathology was observed in animals of the low-concentration group.

The other organs and tissues did not reveal any exposure-related histopathological changes. The histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to the exposure.

Because of the histopathological changes observed in animals of the main groups at the end of the exposure period, microscopic examination of the nasal tissues was extended to animals of the recovery groups (control and high-concentration). Substantial – but not complete – recovery of the nasal lesions was observed at the end of the 4-week exposure-free recovery period. Minimal to mild degeneration of the olfactory epithelium was still observed in 7/10 animals at level 3 and in 1/10 animals at level 4 of the nasal tissues of animals of the high-concentration group. No exposure-related changes were observed in the respiratory epithelium – or in other
parts – of the nasal tissues of animals of the recovery groups.

PLEASE REFER TO THE ATTACHED TABLES - 'Microscopic observations (main and recovery)'
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Bronchoalveolar lavage and measurements
Analysis of BAL parameters at the end of the exposure period revealed an increase in the absolute number of macrophages in BAL fluid of exposed males (and consequently a trend towards increased total / viable cell numbers), which reached the level of statistical significance in males of the high-concentration group. These findings were not associated with any changes in the percentage distribution of white blood cells (BAL fluid normally contains ~100% macrophages in healthy animals). No exposure-related changes in cell differentials were observed in female animals. Investigation of biochemical parameters revealed a slight, but statistically significant increase in gamma-glutamyltransferase (GGT) activity in BAL fluid of females of the mid- and high-concentration group; no changes were found in male animals at the end of the exposure period.

Analysis of BAL parameters at the end of the recovery period did not show any statistically significant differences between exposed animals an unexposed controls, although a trend towards an increased absolute number of macrophages and total cells was still observed in males of the high-concentration group.

PLEASE REFER TO ATTACHED TABLES - 'Bronchiolalveolar lavage: biochemical determinations (main and recovery)' 'Brochiolar lavage: volume and absolute cell count (main and recovery)' 'Bronchiolar lavage: relative cell count (main and recovery)'
Key result
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
49.7 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
10 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no

Estrus cyclicity

Estrus cycle evaluation during the last three weeks of the exposure period did not reveal any exposure-related changes in cycle length or normality of the cycle.

Sperm analysis

Epididymal sperm count

No exposure-related statistically significant differences were observed on epididymal sperm count between the control- and exposure groups.

Epididymal sperm morphology

No exposure-related changes were observed in epididymal sperm morphology. A statistically significantly increased occurrence of ‘small hooks’ in males of the high-concentration recovery group was considered to be a chance finding, because corresponding changes at the end of the exposure period – or any corroborative changes in sperm morphology – were absent.

Epididymal sperm motility

No exposure-related statistically significant differences were observed on epididymal sperm motility. The statistically significantly increased average curvilinear velocity (VCL) in males of the high-concentration recovery group (relative to concurrent controls, but in the same range as controls of the main groups) was considered to be a chance finding, because a similar change was not observed in animals sacrificed at the end of the exposure period (also, a decrease – rather than an increase – in VCL in commonly associated with adverse sperm changes).

Testicular sperm count

There were no statistically significant differences in testicular sperm count parameters between the control- and the high-concentration group.

Analysis of the exposure conditions

Actual concentration

The overall average actual concentrations (± standard deviation) of Dimethylethylamine in the test atmospheres, as determined by total carbon analysis, were 10.0 (± 0.2), 49.7 (± 0.8) and 249.0 (± 4.0) ppm for the low-, mid- and high-concentration groups, respectively. These concentrations were very close to the respective target concentrations of 10, 50 and 250ppm.

Time to attain chamber equilibration (T95)

The time to reach 95% of the steady state concentration (T95), based on chamber volume and the average total flow, was calculated to be 5.1 – 5.7 minutes.

Generation efficiency

The overall average (± standard deviation) generation efficiency, as calculated from the actual concentration, flows and test material consumption, was 84.0 (± 5.3)%. The efficiency of a vapor test atmosphere generation is commonly close to 100%. The slightly lower (than 100%) efficiency of the current test atmosphere generation was probably related to minor losses of test material due to condensation in the ‘liquid catch’, which was installed after the evaporator to prevent condensation down the line.

Total flow, temperature, relative humidity, oxygen and carbon dioxide concentration

The average (± standard deviation) total flows of test atmosphere were 25.9 (± 0.4), 24.5 (± 0.0), 25.2 (± 0.0) and 27.4 (± 0.2) L/min for the control, low-, mid- and high-concentration groups, respectively.

The average temperature (± standard deviation) was 23.05 (± 0.26), 22.99 (± 0.25), 22.81 (± 0.27) and 23.14 (± 0.30) °C for the control, low-, mid- and high-concentration groups,

respectively (Table 1.3). The average relative humidity during exposure was 48.73 (± 0.92), 49.47 (± 0.90), 46.75 (± 0.80) and 39.02 (± 0.84) % for the control, low-, mid-, and highconcentration groups, respectively.

The oxygen concentration during exposure was in the range of 20.2 – 20.4% (v/v) and the carbon dioxide concentration was in the range of 0.275 – 0.549% (v/v), which was

well within the limits of >19% oxygen and <1% carbon dioxide described in OECD guideline 412.

Conclusions:
Under the conditions of the current study, sub-acute inhalation exposure to Dimethylethylamine at actual concentrations of:
• 249.0 ppm resulted in local toxicity in the upper respiratory tract as characterized by degeneration of the respiratory and olfactory epithelium in the nasal tissues and systemic toxicity, as evidenced by significant decrease in body weight and food consumption;
• 49.7 ppm resulted in no systemic toxicity but presence of adverse local changes in the upper respiratory tract, as characterized by degeneration of the respiratory and olfactory epithelium in the nasal tissues;
• Both in the 49.7 and 249.0 ppm groups, substantial, but not full recovery of the nasal pathology was observed within a 4-week post-exposure recovery period;
• 10 ppm resulted in no systemic or local adverse changes.

Based on these findings, the No-Observed-Adverse-Effect-Concentration (NOAEC) for systemic toxicity upon sub-acute inhalation exposure to Dimethylethylamine in rats was placed at 49.7 ppm, and the NOAEC for local toxicity was placed at 10.0 ppm.
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 November 2018 - 01 July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SAMP160321
- Expiration date of the lot/batch: 17 April 2019


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a refrigerator (2 to 8°C), desiccated, in the dark.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:WI(Han) strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: yes]
- Age at study initiation: 53 to 59 days
- Weight at study initiation: Males :222 to 318 g, Females: 143 to 203 g
- Fasting period before study: N/A
- Housing: Cages-Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. The cages constituting each group were blocked together by sex on separate batteries. Five of the same sex per cage (main study and recovery), unless reduced by mortality or isolation. Wood based bedding which was changed at appropriate intervals each week.
- Diet (e.g. ad libitum): Teklad 2014C Diet. Non-restricted (removed overnight before blood sampling for hematology or blood chemistry and during the period exposure).
- Water (e.g. ad libitum): Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Non-restricted (except during exposure).
- Acclimation period: 11 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%.
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

IN-LIFE DATES (Main study): From: 08 November 2018 To: 18 to 19 February 2019
IN-LIFE DATES (Recovery study): From: 08 November 2018 To: 01 April 2019
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Details on inhalation exposure:
Route: Inhalation - snout only exposure.
Control (Group 1): Air only.
Duration of daily exposure: 6 hours. Weeks 1 to 12: 5 days each week; Week 13: 7 days.
Training for dosing: The animals on study were acclimated to the method of restraint, over a 3 day period immediately preceding the first test item exposure.

Exposure System:
Flow through nose-only chamber
Aluminum alloy construction comprising a base unit, three animal exposure sections, a top section and a pre-chamber

Animal Restraint:
Plastic nose-only restraint tube

Atmosphere Generation:
Glass sintered vaporizer
The test item was supplied to the generator, via a feed line, from a syringe driven at a constant rate by a syringe pump

Inlet Airflow:
From in-house compressed air system – breathing quality
Generator flow: 10-60 L/minute

Extract Airflow:
Drawn by in-house vacuum system
Filtered locally
Extract flow: 40-160 L/minute

Airflow Monitoring:
High quality tapered tube flowmeters - calibrated daily
In-line flowmeters monitored continuously

System Containment:
Systems housed in separate ventilated cabinets.

Administration:
Test group animals (Groups 2 – 4) were exposed to an atmosphere containing dimethylethylamine.
Group 1 animals were exposed to compressed air only
Animals were exposed on five days each week for 13 weeks. Additional animal exposures were conducted on Week/Day 13.6, 13.7 and 14.1 to cover end of study
investigations.
Duration of exposure was 6 hours each day
Exposures commenced on 19 November 2018
Different exposure levels were achieved by varying the concentration of test item in the exposure systems, whilst keeping the duration of exposure constant
The animals on study were acclimatized to the method of restraint for three consecutive days preceding their first exposure
System operating conditions were amended at the discretion of the Study Director to maintain achieved atmosphere concentrations close to target.

Concentration:
Atmosphere samples collected as follows:
Collection media: Dreschel head and solvent trap (bubbler)
Sample solvent: Methanol
Sample flow: 2.0 L/minute
Sample volume: Measured by wet-type gas meter
Sample frequency: 1 sample from Group 1/day (taken at approximately 180 minutes into exposure)
Minimum of 3 samples from Group 2, 3 and 4/day (taken at approximately 60, 180 and 300 minutes during exposure)
Sample location: Animal exposure port
Sample analysis: Chemical
During preliminary characterization trials an assessment was made of the percentage breakthrough of test item through the sample collection media; this was achieved by setting up two bubblers in series and collecting a sample of test atmosphere. The acceptable breakthrough limit to the second solvent trap is = 10%. The percentage break through the sample collection media was less than 10%, therefore one bubbler was used on study to collect chamber atmosphere samples.

Chamber air temperature was measured throughout exposure using an electronic thermometer probe placed in the breathing zone of the animals via an unused exposure port. Chamber air temperature was monitored continuously and recorded at 60-minute intervals.

Chamber relative humidity was measured throughout exposure using an electronic hygrometer probe placed in the breathing zone of the animals via an unused exposure port.
Chamber relative humidity was monitored continuously and recorded at 60-minute intervals.

The mean achieved atmosphere concentrations were 103, 100 and 106% of target for Groups 2, 3 and 4, respectively. Initially the inter and intra exposure variation was higher than anticipated. Bubbles were observed to be forming in the syringes and feed lines containing the test item. This was attributed to the test item expanding as it warmed following refrigerated storage and was
remedied by allowing the test item to warm to ambient temperature prior to generation. Subsequently the test item was observed to be vaporizing in the feed lines with an inversely
proportional relationship to target atmosphere concentration. This was considered to be a consequence of the very low feed rates required to achieve the target atmosphere concentrations and was remedied by reducing the diameter of the feed lines.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 Weeks.
Frequency of treatment:
Dimethylethylamine was administered to Wistar Han rats by snout-only inhalation exposure for 6 hours per day (5 days per week) for 13 weeks.
Dose / conc.:
0 ppm
Dose / conc.:
10 ppm (nominal)
Remarks:
Analytical value: 10.3 ppm
Dose / conc.:
30 ppm (nominal)
Remarks:
Analytical value: 29.9 ppm
Dose / conc.:
100 ppm (nominal)
Remarks:
Analytical value: 106 ppm
No. of animals per sex per dose:
10 animals, per sex, per dose + 10 animals per sex for the recovery groups (control and high dose)
Control animals:
yes
Details on study design:
The target doses used in this study (0, 10, 30 and 100 ppm) were selected based on the results of a previous 28-day inhalation study:
After 28 days of exposure to 50 and 250 ppm, degeneration of respiratory and olfactory epithelium in the nose was observed. At 250 ppm, up to moderate degeneration was observed in all animals in all nasal sections with the exception of level 1. Additionally, reduced body weight (almost 10% in males) and reduced food consumption was recorded at 250 ppm. No changes were caused by 10 ppm. Due to the severity of the corrosion of the nasal tissue and body weight differences of almost 10%, 250 ppm was considered to high for a 90-day exposure. Consequently, a high exposure level of 100 ppm was selected. Based on the results obtained at 50 ppm, local effects were still expected in the nose but it was considered that these would be tolerable. Intermediate and low exposure levels of 30 or 10 ppm were selected to assess any relationship to exposure level.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
Cages were inspected daily for evidence of animal ill-health amongst the occupants. Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
Signs Associated with Dosing
Daily during the first four weeks of treatment on exposure days and weekly thereafter, detailed observations were recorded at the following times in relation to dose administration:
Pre-exposure observation
As each animal was returned to its home cage
As late as possible in the working day

In addition observations were made in the treatment period, on days without exposures during Weeks 1 to 4, at the following times during the day:
Early in the working day (equivalent to pre-exposure observation)
As late as possible in the working day

Observations during exposure is severely restricted due to tube restraint.

Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

BODY WEIGHT: Yes
The weight of each animal was recorded twice weekly from one week before treatment commenced, on the day that treatment commenced (Day 1) and during Weeks 1 to 4. Weekly body weights were recorded during Weeks 5 to 13, during recovery and on the day of necropsy.

FOOD EFFICIENCY: Not specified

FOOD CONSUMPTION
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of all animals were examined by means of a binocular indirect ophthalmoscope during pretreatment and Week 13.

HAEMATOLOGY: Yes
Blood samples were collected after overnight withdrawal of food duing week 13 from all animals.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
Hematocrit (Hct)*
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)*
Mean cell hemoglobin concentration (MCHC)*
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)

*Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate. Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
Prothrombin time (PT) - using IL PT Fibrinogen reagent. Activated partial thromboplastin time (APTT) - using IL APTT reagent.

BLOOD CHEMISTRY: Yes
Blood samples were collected after overnight withdrawal of food duing week 13 from all animals.
Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Urea*
Blood urea nitrogen (BUN)
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

*Numerically equivalent to blood urea nitrogen (BUN)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Yes
Sensory reactivity and grip strength assessments were performed (on non-dosing days) on all main study animals in Groups 2 and 3 and all recovery phase animals during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.
The following measurements, reflexes and responses were recorded:

Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers).
Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded.
Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed.
Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded.
Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.

Motor Activity
During Week 12 of treatment (on non-dosing days), the motor activity of all main study animals in Groups 2 and 3 and all recovery phase animals was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.


IMMUNOLOGY: Not specified

OTHER:
Estrous Cycles – Vaginal Smears: Dry smears were taken For 14 days during Weeks 12 and 13 of treatment and during the recovery phase, using cotton swabs.

Mortality: A viability check was performed near the start and end of each working day. Animals were isolated or killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases.

All observations regarding Thyroid hormone analysis please see materials and methods section below.
Sacrifice and pathology:
All main study and recovery animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were checked before disposal of the carcass.
Other examinations:
Bone Marrow
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment or recovery periods and from animals killed prematurely during the study

Bronchoalveolar Lavage (BAL)
The right lung was used for bronchoalveolar lavage sampling and the left lung was processed for histology and light microscopy.

Sperm Analysis
Immediately after scheduled sacrifice of each male and collection of blood and bone marrow, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.
The following tests were performed:
Sperm motility – all groups
Sperm morphology – Groups 1 and 4
Sperm count – all groups
Homogenisation-resistant spermatid count – all groups

Stage-dependent Evaluation of Spermatogenesis
Stage dependent evaluation of spermatogenesis was conducted on sections of testes from all animals of Groups 1 (Control) and 4 (106 ppm) sacrificed on completion of the scheduled treatment period prepared and stained using the PAS method. A qualitative examination of spermatogenic stages was made for normal progression of the stages of the spermatogenic cycle, cell associations, and proportions expected to be present during normal spermatogenesis.



Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related clinical signs or dosing observations during the 13 weeks of treatment or during the 6 week recovery period.
Signs associated with the administration procedure included wet fur and/or red staining of the head, nose and eyes on return to home cage, in which the majority had resolved by end of working day. These signs were seen in animals from all groups including control, therefore are considered to be due to the method and duration of restraint and are commonly seen on inhalation studies of this study design. There were no test-item related effects observed during the physical examination and arena observations.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was one unscheduled death. A Group 2 male, number 20, died under anaesthetic during blood sample collection for hematology and blood chemistry during Week 13. The reason for death is unknown as the animal was considered normal prior to induction of anaesthesia and no macroscopic abnormalities were seen at necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
After thirteen weeks of treatment, group mean body weight gain was lower than control for both sexes exposed to 106 ppm (0.77X and 0.79X control, males and females respectively). Differences in body weight did not reach statistical significance.
There were no test-item related effects on body weight (gain) for either sex exposed to 10.3 or 29.9 ppm.
After 6 weeks of recovery, group mean body weight gain was higher than control for males previously exposed to 106 ppm (1.36X control), while high-dose females gained weight comparable to the controls.
Since effects were transient and body weight gain reached or exceeded the control values quickly after the end of the exposure period, this change was likely secondary to the nasal irritation / stress. It was therefor considered treatment-related but non-adverse.

Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no significant differences in food consumption after 13 weeks of treatment or 6 weeks of recovery.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There was a higher incidence of superficial opacities in males that received 106 ppm after 13 weeks of treatment, evaluation of animals in groups that received 29.9 or 10.3 ppm did not reveal a similar effect. This finding is considered incidental in absence of a similar effect in females or animals in the lower exposure levels.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on haematology.
All differences from control were minor, lacked exposure relationship or were inconsistent between the sexes. Therefore, these were considered to be due to individual variation and unrelated to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on blood chemistry.
All differences from control were minor, lacked exposure relationship or were inconsistent between the sexes. Therefore, these were considered to be due to individual variation and unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects.
Group mean hindlimb grip strength was higher than control for all treated groups (not exposure related), however all were within the range of the historical data therefore this was considered incidental. A small number of differences attained statistical significance, however these were isolated and are attributed to normal variation.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects.
All differences from control were minor, lacked exposure relationship or were inconsistent between the sexes. Therefore, these were considered to be due to individual variation and unrelated to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No differences between control and treated animals were observed after 13 weeks or the additional 6 week recovery period.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Animals Killed After 13 Weeks of Treatment: Changes related to treatment with the test item were seen in the nose/turbinates. Degeneration/atrophy of the olfactory epithelium mainly affecting the dorsal parts of the nasal vestibules was observed in animals receiving 29.9 or 106 ppm and was associated with loss of axon bundles in the sub adjacent lamina propria. Incidence and severity of these changes showed an exposure level response.

Animals Killed After 6 Weeks of Recovery: Minimal degeneration/atrophy of the olfactory epithelium associated with loss of axon bundles in the sub adjacent lamina propria was observed in animals previously exposed to 106 ppm. These changes were mainly distributed in the dorsal part of the nasal cavities.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Estrus cycle: When compared with control, estrus cycles in females exposed to 106 ppm during Weeks 12 and 13 showed a higher number of irregular cycles. Of the 20 animals, 5 had irregular cycles versus 1 control animal. There were also two individuals exposed to 106 ppm that either had extended estrus or were determined to be acyclic. During the recovery period, there was one control female with an irregular cycle and extended estrus for two females previously exposed to 106 ppm. Since a similar incidence was also observed in the low dose (2 (out of 10) low dose females showed an irregular cycle and 1 additional female had an extended estrus), while no mid-dose females showerd irregularities (lack of dose-response), and because a similar incidence of divergence was also observed in one of the recent historical control studies, the changes in the high-dose were considered as incidental.

T3/T4 analysis: There were no test item-related effects. All samples taken from all groups, including control, at termination and from control and animals previously exposed to 106 ppm at the end of the recovery phase showed T3 and T4 concentrations were consistent among groups.

Thyroid Stimulating Hormone analysis: Individual serum TSH concentrations were found to be variable. Group mean TSH concentrations were lower for males exposed to 10.3 ppm when compared with control. Group mean TSH concentrations for males exposed to 29.9 or 106 ppm were similar to control. There was a slight increase in TSH concentrations for males previously exposed to 106 ppm when compared with control. Females showed an increase in TSH concentrations with increasing concentration of Dimethylethylamine when compared with control. There was a slight decrease in TSH concentrations for females previously exposed to 106 ppm when compared with control.
Given the high degree of variability, lack of exposure related response in males and inconsistency between the sexes, it is considered the observed changes are likely to be a result of biological variation rather than a test-item related effect.

Sperm Analysis: No adverse effects on sperm motility, testicular spermatid numbers, cauda epididymal sperm numbers or sperm morphology were observed following treatment with Dimethylethylamine compared with control.

Bronchoalveolar Lavage (BAL): There were no test item-related effects.
Group mean cell counts were variable when compared with control, however individual values for test animals were within the control range and therefore all differences were attributed to normal biological variation.

Total Protein and Lactate Dehydrogenase: Although group mean data may suggest lower total protein and lactate dehydrogenase concentrations in treated males and higher total protein and lactate dehydrogenase concentrations in females, there was a large degree of variation and overlap in individual data when comparing test data with control, so there is no convincing test item-related effect.
After 6 weeks of recovery, higher group mean total protein (up to 1.82X control) and lactate dehydrogenase concentrations (up to 1.64X control) were observed in both sexes exposed to 106 ppm when compared with control.
Dose descriptor:
NOAEC
Remarks:
nasal local effects
Effect level:
10.3 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other:
Remarks:
31 mg/m3
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
>= 106 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
318 mg/m3
Critical effects observed:
no

Summary of treatment related findings in the nose/turbinates for animals killed after
13 weeks of treatment
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Exposure level (ppm) 0 10.3 29.9 106 0 10.3 29.9 106
Degeneration/Atrophy, Olfactory
Epithelium with Loss of axon Bundles
               
Minimal 0 0 2 3 0 0 4 1
Slight 0 0 0 6 0 0 0 5
Moderate 0 0 0 1 0 0 0 4
Total 0 0 2 10 0 0 4 10
Number of tissues examined 10 10 10 10 10 10 10 10

Summary of treatment related findings in the nose/turbinates for animals killed after
6 weeks of recovery
Group/sex 1M 4M 1F 4F
Exposure level (ppm) 0 106 0 106
Degeneration/Atrophy, Olfactory
Epithelium with Loss of axon Bundles
       
Minimal 0 5 0 4
Slight 0 3 0 0
Total 0 8 0 4
Number of tissues examined 10 10 10 10
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
A detailled read across justification has been attached to IUCLID chapter 13.
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
>= 106 ppm
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
10.3 ppm
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
125 mg/m³
Study duration:
subacute
Species:
rat

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

To evaluate the toxicity profile of the test substance after inhalation exposure, groups of 10 male and 10 female Wistar rats (F0 animals) per test group were exposed in a whole-body inhalation system to dynamic atmosphere for 6 hours per day on each day in a GLP-compliant OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test (2019). The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 6 days postmating in males, and the gestation (up to GD 19) and the lactation period in females from PND 4 onwards up to the day of scheduled sacrifice of the animals. Pups of the respective dams were exposed from PND 4 until PND 13. The target concentrations were 125 mg/m³ (35 ppm), 500 mg/m³ (140 ppm) and 1500 mg/m³ (420 ppm). A concurrent control group was exposed to conditioned air. The NOAEC (no observed adverse effect concentration) for local toxicity of the test substance in the respiratory tract was the lowest tested concentration of 125 mg/m³ based on adverse effects such as degeneration/regeneration of the olfactory epithelium in the nasal cavity of the parental animals after inhalation (vapor) exposure to 500 and 1500 mg/m³. The NOAEC for systemic toxicity was the mid concentration of 500 mg/m³ as adverse clinical signs and a reduction in food consumption together with a decrease in body weight (change) occurred in both sexes at the highest concentration of 1500 mg/m³. The NOAEC for reproductive performance and fertility of the F0 parental rats and developmental toxicity in the offspring was the highest tested concentration of 1500 mg/m³.

 

For the structurally related substance DMEA (598 -56 -1), two further repeated dose toxicity studies are available.

Rats were exposed nose only for 28 days to concentrations of 10, 50, and 250ppm in a study according to OECD 412. The highest dose led to reduced body weight associated with a decrease in food consumption. Degeneration of the olfactory and respiratory epithelium in the upper respiratory tract occured dose-dependently in mid and high dose animals. There was substantial though not full recovery after 4 weeks without treatment. The NOAEC for systemic toxicity was set at 50 ppm, the NOAEC for local toxicity was 10 ppm in this study.

In a study according to OECD 413, DMEA was administered by snout-only inhalation to Wistar Han rats for 6 hours a day, 5 days a week, for 13 weeks. Achieved exposure levels were 10.3, 29.9 or 106 ppm, which were clinically well tolerated. Recovery was assessed during a 6 week off-dose period. Test item-related changes were evident in the nasal turbinates of animals exposed to 29.9 or 106 ppm and consisted of minimal to moderate degeneration/atrophy of the olfactory epithelium mainly affecting the dorsal parts of the nasal vestibules and was associated with loss of axon bundles in the sub adjacent lamina propria.  There was evidence of partial recovery after 6 weeks without exposure to DMEA. Based on these findings, a local NOEAC of 10.3 ppm was determined. No systemic toxicity occured.

Based on the available combined repeated dose and reproductive/developmental toxicity screening study (OECD TG 422, 2019) with DMPA and data of the read across substance DMEA, local irritant effects are considered the leading health effect. There is no indication for systemic repeated dose toxicity in addition to local toxicity at the point of entry. General systemic toxicity is likely a result of stress and local irritation of the upper respiratory tract.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result, the substance is not classified for repeated dose toxicity according to Regulation (EC) No 1272/2008.