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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Inhalation

- OECD 422; GLP; 2019; 10 Wistar rats/sex/dose; 125, 500, and 1500 mg/m³; NOAEC (repro) 1500 mg/m³ (highest dose tested), NOAEC systemic = 500mg/m³, NOAEC local = 125mg/m³

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb 2018 - May 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
Details on species / strain selection:
Wistar Rat; Crl:WI(Han)
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;
- Fasting period before study: not specified
- Housing: 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, see 3.7.4. “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 (e.g. ad libitum): Kliba maintenance diet mouse/rat “GLP” meal, supplied by Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland ad libizum.
- Water (e.g. ad libitum):Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland, 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 (if applicable):
whole body
Vehicle:
air
Details on 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, pressure in air chamber: range of temperature of 20-24°C and a range of relative humidity of 45-65%.
- 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 vapor / 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.

In order to accustom the animals to the exposure conditions they were placed into exposure cages before start of the exposure period on study days -5, -4 and -1 (preexposure period) (details are available in the raw data).
Then all test groups were exposed for 6 hours on each day per week for the following period of time:
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.
Exposure period:
Test groups 0 - 3: A positive pressure was maintained by adjusting the air flow of the exhaust air system.
For same exposure conditions, the cages with the animals were rotated between the levels within each chamber.
Details on mating procedure:
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".
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.
Frequency of treatment:
6 hours/ day, every day
Details on study schedule:
Study initiation date: 06 Feb 2018
Experimental starting date (arrival of the animals): 06 Feb 2018
Randomization date: 26 Feb 2018
Acclimatization period: 06 Feb - 05 Mar 2018
Pre-exposure period: 01 - 05 Mar 2018
Exposure period: 06 Mar - 03 May 2018
Mating period: 19 - 24 Mar 2018
Gestation period: 20 Mar - 14 Apr 2018
Parturition: 11 - 15 Apr 2018
Lactation period: 11 - 28 Apr 2018
FOB and MA: F0M: 05 Apr 2018; F0F: 02 May 2018
Blood sampling: F0M: 09 Apr 2018; F0F: 25 - 29 Apr 2018
Sacrifice of litters: 24 - 28 Apr 2018
Sacrifice of parental animals: F0M: 09 Apr 2018; F0F: 04 May 2018
Experimental completion date: 22 May 2019
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.
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".

Parental animals: Observations and examinations:
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 tthe OECD Guidlines for testing of Chemicals No.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 OECD Guidlines for testing of Chemicals No.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 OECD Guidlines for testing of Chemicals No.422(2016).
The implantations were counted1) and the postimplantation loss (in %) was calculated according to the OECD Guidlines for testing of Chemicals No.422(2016).

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, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume,Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, differential blood count, Reticulocytes Furthermore: prothrombin time.
Clinical chemistry
An automatic analyzer (Cobas c501; Roche, Mannheim, Germany) was used to examine the following clinicochemical parameters: ALT, AST, ALP, GGT, Na, K, Cl, inorganic Phosphate, 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.
Oestrous cyclicity (parental animals):
For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization (the estrous cycle data o these individuals were not be reported and can be found in the raw data). For a minimum of 2 weeks prior to mating estrous cycle length 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.
Litter observations:
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, sex ratio, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups
- mortality/moribund: yes, twice daily
- Viability index (%) = (No of live pups on day 4 after birth) / (No of live pups on day of birth) * 100
- Survival index (%) = (No of live pups on day 13 after birth) / (No of live pups on day 4 after birth) * 100

GROSS EXAMINATION OF DEAD PUPS:
- yes, after sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically.
- thyroid hormone examination on PND 4 (surplus pups) and PND 13 (one male and one female)


ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no
Postmortem examinations (parental animals):
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, Seminal vesicles with coagulating glands, Testes, Thyroid 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, Brain, Heart, Kidneys, Liver, 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 according to the table attached ("Histopathology").
Special attention was given to the stages of spermatogenesis in the testes 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.
Postmortem examinations (offspring):
SACRIFICE
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
- On PND 4, surplus pups were sacrificed and blood was sampled for determination of thyroid hormone concentrations
- On PND 13, one selected male and one female pup per litter was sacrificed and blood was sampled for determination of thyroid hormone concentration (Thyroid glands/parathyroid glands were fixed)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGTHS
- only animals with notable findings, depending on the type of the finding
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.
Reproductive indices:
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 to the following formulas:
Male mating index (%) = (number of males with confirmed mating / number of males placed with females) x 100
Male fertility index (%) = (number of males proving their fertility / number of males placed with females) x 100

Female reproduction and delivery data
The pairing partners, the number of mating days until vaginal sperm were detected and gestational status were recorded for F0 females.
For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:
Female mating index (%) = (number of females mated / number of females placed with males) x 100
Female fertility index (%) = (number of females pregnant / number of females mated) x 100
Gestation index (%) = (number of females with live pups on the day of birth / number of females pregnant) x 100
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:
Live birth index (%) = (number of liveborn pups at birth / total number of pups born) x 100
The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:
Postimplantation loss (%) = ((number of implantations – number of pups delivered) / number of implantations) x 100

To determine the number of implantation sites, the apparently non-pregnant uteri were stained for about 5 minutes in 1% ammonium sulfide solution according to the method of Salewski, E.; 1964. The implantation sites were recorded for calculation of the postimplantation loss.
Offspring viability indices:
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PNDs 1-4, 5-7 and 8-13 were determined. Pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day of birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated according to the following formulas:
Viability index (%) = (number of live pups on day 4 after birth / number of live pups on the day of birth) x 100
Survival index (%) = (number of live pups on day 13 after birth / number of live pups on day 4 after birth) x 100
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.
Dermal irritation (if dermal study):
not examined
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 the whole 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 3males 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 test substance-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
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.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data generated during the last 2 weeks prior to mating for the F1 litter revealed regular cycles in the females of all test groups (0, 125, 500 or 1500 mg/m³). The mean estrous cycle duration was 3.9 / 4.1 / 4.0 / 5.3* (p ≤ 0.05) days in test groups 0-3, respectively. The statistically significantly increased value in test group 3 was caused by one single test group 3 female (animal No. 132) which had a prolonged mean cycle length of 12.00 days. This increased cycle length of female No.132 led to a higher standard deviation value of test group 3 compared to control (2.4 vs 0.2). All other females of test group 3 had cycles between 3.7 and 5.5 days. This range is comparable to the other test groups and the control. Thus, the lower mean of test group 3 females was solely caused by the single outlier and is therefore assessed as not treatment-related.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Male reproduction data
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in test groups 0-3. Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
One control male (0 mg/m³ - No. 4) did not generate pregnancy. Thus, the male fertility index ranged between 90% and 100% without showing any relation to´dosing. This reflects the normal range of biological variation inherent in the strain of rats used for this study.
The apparently infertile male rat did not show relevant gross and histopathological lesions

Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm wasdetected (GD 0) varied between 2.5 and 2.8 days without any relation to dosing. All female rats delivered pups or had implants in utero with the following exception: Control female No. 104 (mated with male No. 4) did not become pregnant.The fertility index varied between100% in test groups 1, 2 and 3 and 90% in test group 0. These values reflect the normal range of biological variation inherent in the strain of rats used for this study.
The non-pregnant female had no relevant gross and histopathological lesions. The mean duration of gestation was similar in all test groups (i.e. between 22.1 and 22.3 days). The gestation index varied between 100% in test groups 0, 1 and 3 and 90% in test group 2. One mid-concentration female (No. 127 - 500 mg/m³) had 7 pups and all pups were stillborn. Since it was only one dam affected without any relation to concentration, it was assessed as not treatment-related and not adverse. Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.4 / 12.7 / 13.3 a d 11.9 implants/dam in test groups 0-3, respectively).
Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (11.9 / 12.4 / 11.8 and 11.1 pups/dam in test groups 0-3, respectively). The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 100.0 / 97.6 / 90.7 and 99.1% in test groups 0-3. Moreover, the number of stillborn pups was not significantly different between the test groups. Thus, the test substance did not adversely affect reproduction and delivery of the F0 generation parental females.
Under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats, 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.
Key result
Dose descriptor:
NOAEC
Remarks:
reproductive toxicity
Effect level:
1 500 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
500 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
food consumption and compound intake
Dose descriptor:
NOAEC
Remarks:
local
Effect level:
125 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
There was no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The pup mortality is based on total number of stillborn pups, dead pups, pups sacrificed moribund and cannibalized pups.
The viability index indicating pup survival during early lactation (PND 0-4) varied between 98.4% / 95.7% / 97.8% and 96.9% in test groups 0-3, respectively, without showing any association to treatment.
The pups surviving index indicating pup survival during remaining lactation (PND 4-13) was 100% in all test groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
One female runt was seen in test group 2.
Mean body weights of male and female F1 pups of test group 3 (1500 mg/m³) were statistically significantly reduced on the day of scheduled sacrifice on PND 13 (20% below control, both sexes combined).The mean pup body weight changes of the high-concentration F1 pups were statistically significantly below the concurrent control values during PND 4-13 (up to -36%) and if calculated for the entire lactation period (PND 1-13, -26%, both sexes combined). Up to PND 4, body weight development was not affected in any of the test groups showing comparable mean body weights during early lactation. With start of inhalation exposure from PND 4 onwards, a delayed body weight development of test group 3 pups was observed and assessed as secondary to the direct exposure of the pups to the test substance. It was not assessed as an independent developmental effect. In test group 2 the mean pup body weight changes were statistically significantly reduced on PND 7-13 (12.7 g vs 14.4 g in control, both sexes combined). However, if calculated for the entire lactation period, mean pup body weight changes were comparable tothe concurrent control value. Since the decrease occurred only during one short time period, it was not assessed as treatment-related and adverse. No test substance-related influence on pup body weights were noted in test groups 1 and 2 (125 and 500 mg/m³) and on pup body weight change in test group 1.
Food consumption and compound intake (if feeding study):
not examined
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
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A few pups showed spontaneous findings at gross necropsy, such as hydroureter, dilated renal pelvis, hydronephrosis, fluid-filled abdomen and post mortem autolysis.
These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Mean number of delivered F1 pups per dam and rates of liveborn, stillborn, cannibalized and dead F1 pups were evenly distributed
- Sex ratio: no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
1 500 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed up to high dose
Critical effects observed:
no
Reproductive 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.
Effect on fertility: via oral route
Study duration:
subacute
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 500 mg/m³
Experimental exposure time per week (hours/week):
42
Species:
rat
Effect on fertility: via dermal route
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 post-mating 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 (34 ppm1), 500 mg/m³ (138 ppm1) and 1500 mg/m³ (414 ppm1). 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.

Based on the available combined repeated dose and reproductive/developmental toxicity screening study (OECD TG 422, 2019) with DMPA local irritant effects are considered the leading health effect. There is no indication for reproductive toxicity.

Further, the suitable structural analogue substance, Dimethylethylamine CAS 598-56-1, is currently under intensive investigation. The results of the studies on toxicity to reproduction will be available in the near future. Once the results become available, the dossier will be updated accordingly. The justification for this read across approach has been attached in chapter 13.

Effects on developmental toxicity

Description of key information

Oral:

- OECD 414; GLP; 2020; 20 Sprague-Dawley rats/sex/dose; 20, 60, and 180 mg/mkg/bw; NOAEL (mat.) = 60 mg/kg/bw, NOAEL (dev.) = 180 mg/kg/bw

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Source and lot/batch number of test material: SAMP18373
- Purity. 99.57%
Species:
rat
Strain:
Sprague-Dawley
Remarks:
[Crl:CD(SD)]
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: 70 to 76 days old
- Weight at study initiation: 210 to 293 g
- Housing: Acclimatization - up to four animals; During pairing - one (stock) male and one female; Gestation - one female
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet (ad libitum)
- Water (e.g. ad libitum): tap water (ad libitum)
- Acclimation period: Six days before commencement of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
purified water
Analytical verification of doses or concentrations:
yes
Details on mating procedure:
Male/female ratio: 1:1 with identified stock males.
Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation: When positive evidence of mating was detected.
Duration of treatment / exposure:
GD 6-19
Frequency of treatment:
once daily
Duration of test:
20 d
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
60 mg/kg bw/day (nominal)
Dose / conc.:
180 mg/kg bw/day (nominal)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The high dose level of 180 mg/kg/day was selected based on the results of a preliminary embryo-fetal study in the female rat conducted at these laboratories.
In that study animals received daily administration of Ethyldimethylamine over Days 6-19 of gestation at 50, 180 and 360 mg/kg/day.

Females receiving 360 mg/kg/day started showing signs of decreased activity, partially closed eyelids, flattened or hunched posture, shallow breathing, cold to touch and piloerection on Day 8 or 9 of gestation. Two females were euthanised for welfare reasons, both on Day 9 of gestation, after more than 15% bodyweight loss, which reached or exceed the moderate severity limit. The high dose level was
subsequently reduced to 270 mg/kg/day for the remaining four females. These females were prostrate at 30 minutes post dosing and underactive, hunched with piloerection and had abnormal breathing at 1 hour post dose. Prior to dosing on Day 7 of gestation at the reduced high dose, one female was found dead and a second female had lost body weight, exceeding the moderate severity limit. As the dose level
was not tolerated, the remaining females were euthanised for welfare reasons on Day 9 or 10 of gestation.

Treatment at 180 mg/kg/day was tolerated, but was associated with reduced weight gain (86% of Controls), reduced adjusted maternal weight gain (60% of Controls) and a slight reduction in food consumption (90% of Controls). One female had depressions on the nonglandular region of the stomach but remained in good clinical condition.

Therefore a high dose of 180 mg/kg/day was considered suitable as the high dose with 60 and 20 mg/kg/day chosen as the intermediate and low dose levels to allow assessment of any dose response.

Maternal examinations:
Clinical Observations:
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing:
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:
Pre-dose observation. One to two hours after completion of dosing. As late as possible in the working day.

Clinical Signs:
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

Body Weight:
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

Food Consumption:
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

Necropsy
All adult 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.
Schedule: Animals surviving until the end of the scheduled study period were killed on Day 20 after mating.
Sequence: To allow satisfactory inter-group comparison.
Ovaries and uterine content:
For females surviving to term, the following was recorded:
Uterus: Gravid uterine weight (including cervix and ovaries).
The following were recorded for all animals
For each ovary/uterine horn: Number of: Corpora lutea. Implantation sites. Resorption sites (classified as early or late). Fetuses (live and dead).
For apparently empty uterine horns: The number of uterine implantation sites were checked after staining with ammonium sulphide
Fetal examinations:
Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with
abnormalities recorded. The sex of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.

Fixation: Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.

Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning. IMS fixed fetuses were processed and double stained with Alizarin and Alician Blue.

Fetal Pathology Examination
Bouin’s fixed fetuses: Serial sections were examined for visceral abnormalities.
Double stained with Alizarin Red and Alician Blue fetuses: Assessed for skeletal and cartilage development and abnormalities.
Statistics:
Different test methods were used: Bartlett`s test; Williams`test; Dummet`s test; Kruskal-Wallis`test; Shirley`s test, Steel`s test
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs associated with dose administration were restricted to the animals receiving 180 mg/kg/day, with 4/20 exhibiting a low incidence of rales (no. 61 on one occasion; no. 65 on two occasions; no. 67 on two occasions; no 71 on three occasions) and 4/20 females exhibiting increased salivation each on a single occasion. Signs at routine physical examination were limited to two females at 180 mg/kg/day (nos. 65 and 71) with rales; there were other signs that could be related to treatment. The clinical condition of animals receiving 20 or 60 mg/kg/day was unaffected by treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females (nos 61 and 67) receiving 180 mg/kg/day were killed for welfare reasons; female no 61 was euthanised on GD14 and female no.67 was euthanised on GD12. Terminal signs for both of these females comprised of respiratory distress (gasping) and body weight loss, however no macroscopic abnormality was apparent at necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
From GD9 (Day 4 of treatment) mean body weight gain for females receiving 180 mg/kg/day was low with the difference attaining statistical significance (p<0.01) and overall (GD6-20) the mean body weight change at 180 mg/kg/day was approximately 14% lower than Controls (p<0.01). The resultant mean bodyweight on GD20 for females receiving 180 mg/kg/day was approximately 6% lower than Controls (p<0.05).
The mean gravid uterine weight was essentially similar across the groups. However the maternal weight gain following adjustment for the gravid uterine was statistically significantly low at 180 mg/kg/day, approximately 40% lower than Controls (p<0.01) and the maternal mean weight on GD20 was approximately 6% lower than Controls (p<0.05). Both the absolute body weight gain and adjusted maternal weight gain of females receiving 20 or 60 mg/kg/day was unaffected by treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 180 mg/kg/day mean food consumption was low when compared with Controls with the difference attaining statistical significance for GD10-17 (p<0.01; 86-88% of Controls) and GD18-19 (p<0.05; 93 % of Controls). Overall the total food consumption during the treatment period (GD 6 -19) was approximately 10% lower than Controls.
Food consumption at 20 or 60 mg/kg/day was unaffected by treatment.
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
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic examination of females on GD20 did not reveal any findings that could be attributed to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A higher incidence of post-implantation loss was observed at 180 mg/kg/day (9.3% versus 2.3% in Controls), however the incidence was within the historical control range and there was no impact on the live litter size. This difference was therefore not considered to be related to treatment.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
mortality
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 60 mg/kg/day and 180 mg/kg/day there was a slightly higher incidence of incompletely ossified 1st to 4th sternebrae, compared with concurrent control (12/11 fetuses in 8 litters per group versus 4 fetuses in 4 Control litters); the incidences were within the historical control range of the litters, which is the principal unit of evaluation, with the exception of fetuses at 60 mg/kg/day which exceeded the fetal historical control range. Incomplete ossification is a transient stage in development and is therefore not considered to be adverse.
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
no effects observed
Developmental effects observed:
no
Conclusions:
It was therefore concluded that the no observed adverse effect level (NOAEL) for maternal toxicity was 60 mg/kg/day based on the effect on maternal body weight and food consumption. For embryo-fetal survival and development the NOAEL was considered to be 180 mg/kg/day.
Additional information

Teratogenicity studies according to OECD 414 are performed with the suitable structural analogue substance, Dimethylethylamine CAS 598 -56 -1 (DMEA). The results of the studies on toxicity to reproduction in rabbits will be available in the near future. Once the results become available, the dossier will be updated accordingly. The justification for this read across approach has been attached in chapter 13.

 

OECD 414, rat, DMEA

Three groups of 20 females Sprague-Dawley rats received Ethyldimethylamine at doses of 20, 60 or 180 mg/kg/day by oral (gavage) administration, from Day 6 to 19 after mating. Animals were killed on Day 20 after mating. Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterine weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

 

Two females receiving 180 mg/kg/day were killed for welfare reasons on GD12/14. Terminal signs for both of these females comprised of respiratory distress (gasping) and body weight loss, however no macroscopic abnormality was apparent at necropsy. At the high dose (180 mg/kg/day) there was a low incidence of rales and increased salivation that were attributed to treatment. The high dose animals also exhibited low absolute body weight gain, low food consumption and low maternal weight gain following adjustment for the gravid uterine weight at approximately 86%, 90% and 60% of control animals, respectively. Clinical condition, body weight and food consumption for mid and low dose animals were unaffected by treatment. Macroscopic examination of females at necropsy did not reveal any abnormality that could be related to treatment.

Litter data, fetal and placental weights were unaffected by maternal treatment at dose levels up to and including 180 mg/kg/day. A higher incidence of post-implantation loss at 180 mg/kg/day (9.3% versus 2.3% in Controls) was not considered to be realted to treatment, since the incidence was within the historical control range and there was no impact on the live litter size. The incidence of external, sceletal, and visceral malformation or variants showed no relationship to treatment.

 

It was therefore concluded that the no observed adverse effect level (NOAEL) for maternal toxicity was 60 mg/kg/day based on mortality and reduced maternal body weight and food consumption. For teratogenicity, the NOAEL was considered to be 180 mg/kg/day, the highest dose tested.

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 toxicity to reproduction according to Regulation (EC) No 1272/2008.

Additional information