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EC number: 203-518-7 | CAS number: 107-75-5
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
![](https://echa.europa.eu/o/diss-blank-theme/images/factsheets/A-REACH/factsheet/print_toxicological-information.png)
Endpoint summary
Administrative data
Description of key information
- Oral:
OECD 422, GLP, NOAEL (general systemic toxicity) = 5000 ppm (equivalent to 770 mg/kg bw/d in males and 492 mg/kg bw/d in females) (BASF 2020)
- Inhalation:
OECD 412, GLP, NOAEC = 70 mg/m³ (RIFM, 2013)
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- repeated dose toxicity: oral, other
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 29 July 2016
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Purity: 93.6 corr. area%
- Physical state, appearance: liquid, colorless, clear
- Homogeneity: Given (visual)
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient
- Stability under test conditions: No reanalysis necessary until 11 Dec 2020. The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility. - Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: about 11-12 weeks (male animals); about 10 weeks (female animals)
- Housing: individually
- Diet: ad libitum; ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA (new name Granovit AG), Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 21 days
DETAILS OF FOOD AND WATER QUALITY: The drinking water was regularly assayed for chemical contaminants as well as for the presence of microorganisms. On the basis of the analytical findings the drinking water was found to be suitable. The supplier assayed the food used in the study for chemical and microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12
IN-LIFE DATES: From: 23 Jul 2019 To: 13 Sep 2019 (males); 14 Oct 2019 (females) - Route of administration:
- oral: drinking water
- Details on route of administration:
- The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard.
- Vehicle:
- unchanged (no vehicle)
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Hydroxycitronellal was applied as a solution. To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was weighed out to the desired amount of the test substance preparation and subsequently mixed with a magnetic stirrer until solved. The test substance preparations were produced twice a week, at least. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The analyses of the test-substance preparations were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
The stability of Hydroxycitronellal in drinking water over a period of 7 days at room temperature was proven in a comparable batch (purity 99.6%). It was considered that the stability of the current batch (purity 93.6%) is comparable.
At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 1 sample was taken for the aim to determine the concentration for a concentration control analysis. All test samples, plus a duplicate set of reserve samples, were withdrawn by the staff of the Mechanistic Toxicology. Additional reserve samples were kept frozen (at -20°C). The samples within this study were labeled with serial numbers. The same number will not be used for several samplings. Reserve samples were labeled with 1R, 2R, etc.
Details of the sampling schedule were recorded with the raw data.
The samples collected at the beginning of the administration period and during lactation were analyzed in the Analytical Laboratory. Due to imprecision in one sample of the highest concentration during lactation period the reserve sample was analyzed as well. - Duration of treatment / exposure:
- males: 30 days; females: 62 days
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals. - Frequency of treatment:
- The test substance was administered daily via the drinking water.
During the lactation period the Hydroxycitronellal concentrations in the drinking water of the F0 females were reduced to 50%. This drinking water adjustment derived from historical body weight and water consumption data did maintain the dams at the desired target doses of Hydroxycitronellal during this period of increased water intake. - Dose / conc.:
- 1 500 ppm
- Remarks:
- low-dose level; Test Group 1
- Dose / conc.:
- 5 000 ppm
- Remarks:
- mid-dose level; Test Group 2
- Dose / conc.:
- 15 000 ppm
- Remarks:
- high-dose group; Test Group 3
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- - Dose selection rationale:
A test study (BASF project No. 10C0243/09C159) was performed beforehand to select proper dose levels for the present OECD 422 study. Hydroxycitronellal was administered via the drinking water to groups of 3 female Wistar rats at dose levels of 0 (test group 0), 5000 (test group 1) and 15000 (test group 2) ppm over a period of 2 weeks.
In this test study administration of Hydroxycitronellal via the drinking water to female Wistar rats for 14 days resulted in a decreased water consumption but without correlating effects on the body weight. Therefore, it was considered that the palatability is acceptable for further studies using the administration via the drinking water up to 15000 ppm.
The oral route was selected since this was proven to be suitable for the detection of a toxicological hazard. - Positive control:
- no
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams)
- Time schedule: at least once daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size
BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period (to randomize the animals), study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning) with the following exceptions for the females:
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 4, 7, 10, 14, 17 and 20.
• Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
• Body weight was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: once a week 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 parental animals).
• Food consumption of the females with evidence of sperm was determined for GD 7, 14 and 20.
• Food consumption of the females which gave birth to a litter was determined for PNDs 4, 7, 10 and 13.
- Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.
WATER CONSUMPTION: Yes
- Time schedule for examinations:
Generally, water consumption was determined once a week (over a period of 3 or 4 days) for the male and female parental animals.
The following exceptions are notable for the female parental animals:
• Water consumption of the females with evidence of sperm was determined for GD 0-1, 3-4, 6-7, 9-10, 13-14, 16-17 and 19-20.
• Water consumption of the females which gave birth to a litter was determined for PND 1-2, 3-4, 6-7, 9-10, and 12-13.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.
INTAKE OF TEST SUBSTANCE:
The mean daily intake of test substance (group means) was calculated based upon individual values for body weight and mean water consumption per animal for the different phases of the study. Afterwards, the balanced mean of mean was used to consider the different length of the phases in the study for females (premating, gestation and lactation). Calculation of substance intake for individual phases of the study:
Test substance intake [mg/kg/day] = (water consumption per day [g/day] * nominal dose [mg test item/g vehicle]) / (average body weight during the consumption interval [kg])
For the calculation of balanced mean of mean in females following durations of the phases were used: premating – 13 days, gestation – 20 days, and lactation – 13 days.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 1 were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters checked in table 2 were examined.
URINALYSIS: Yes
- Time schedule for collection of urine: Day 29 (males), Day 59 (females)
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters examined: pH, Protein (PRO), Glucose (GLU), Ketones (KET), Urobilinogen (UBG), Bilirubin (BIL), Blood, Specific gravity (SP.GR.), Sediment, turbidity Color, Vol (VOL)
NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")
OTHER:
Functional observational battery (FOB):
- A functional observational battery was performed in all animals at the end of the administration period starting at about 10.00 h.
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations (at least for 2 minutes): behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (auditory startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings
Motor activity assessment:
- measured from 14:00 h onwards on the same day as the FOB was performed
Estrous cycle:
- In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear. Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.
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.
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.
Thyroid hormones (males only)
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all surviving males at termination
- Parameters checked in table 3 were examined. - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
Animal that were sacrificed moribund were necropsied and assessed by gross pathology as soon as possible after their death.
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Epididymides
3. Ovaries
4. Prostate (ventral and dorsolateral part together, fixed)
5. Seminal vesicles with coagulating glands (fixed)
6. Testes
7. Thyroid glands (with parathyroid glands) (fixed)
8. 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):
1. Adrenal glands (fixed)
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus (fixed)
All paired organs were weighed together (left and right).
HISTOPATHOLOGY: Yes (see table 4)
Animals that died or were sacrificed in a moribund state were processed histotechnically and assessed like control animals. - Statistics:
- see table 5
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- In one male animal of test group 3 (No. 38; 15000 ppm) a slightly poor general condition and piloerection was recorded during detailed clinical observation on study day 7. On the same study day piloerection was recorded in 4 female animals of test group 3 (Nos. 134, 135, 136 and 138; 15000 ppm). One of those females (No. 138) even showed closed eyelids, a slightly poor general condition, piloerection and smeared fur on this day. One female (No. 135) did not nurse its pups properly on lactation day 0.
All clinical observations in the moribund animals Nos. 38 and 138 in test group 3 were assessed as treatment-related and adverse. The clinical findings observed in the other animals of this test group were considered as treatment-related but not adverse because they have been observed only on a single study day. - Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Male animal No. 38 and female animal No. 138 of test group 3 (15000 ppm) were sacrificed moribund on premating day 8. Prior to and as a reason for that the following clinical signs were observed: A slightly poor general condition was first observed in animal No. 138 on premating days 5 to 7 and in animal No. 38 on day 7. Furthermore female No. 138 showed closed eyelids and brownish smeared fur on its forelimbs from day 5 onwards, piloerection on day 4 and from day 6 onwards, and on day 8 general condition was severely poor and a severe reduced nutritional condition was observed. Male No. 38 showed piloerection from day 7 to 8, as well as closed eyelids, severely poor general condition and moderate reduced nutritional condition on day 8. The cause for the observed findings in these two animals could not be determined after pathological examinations and no other animal died ahead of schedule. Thus, after completion of all related examinations, the cause of the clinical moribund status could not be identified. Its occurrence in the high dose group (15000 ppm) was as related to treatment and adverse.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Body weights were significantly reduced in male animals of test group 3 (15000 ppm) from day 7 onwards with a maximum of -7.6% (day 7) and in female animals of test group 3 (15000 ppm) from gestation day 10 onwards with a maximum of -19.6% (GD 20). The significantly reduced body weight during gestation could have been caused by 6 animals without delivered pups and a therefore lower body weight compared to the animals with delivery. During lactation period the two female animals with litter (Nos. 135 and 139) of test group 3 (7500 ppm) as well showed reduced body weights with a maximum of -8.1% compared to control group.
Body weight changes were also reduced significantly in males of test group 3 (15000 ppm). A body weight loss was recorded from study day 0 to 7 (-20.6g) and reduced body weight gain from day 7 to 13. Due to these low values the overall body weight gain (day 0 to 28) was significantly reduced as well. Female animals of test group 3 (15000 ppm) showed significantly reduced body weight gain as well. A body weight loss was recorded from study day 0 to 7 (pre-mating, -8.5 g) and therefore the body weight change of the overall interval of pre-mating period (day 0 to 13) was significantly reduced. During gestation a significantly
reduced body weight gain was recorded from gestation day 7 onwards and for the overall interval of gestation (gestation day 0 to 20). The latter finding could be caused by 6 females without delivery in this test group. The two females with litter of test group 3 (7500 ppm) also showed a reduced body weight gain during lactation period (lactation day 1 to 13) as well as a body weight loss from lactation day 10 to 13 (-3.5g).
These findings in mean body weight and body weight changes of males and females in test group 3 (15000 / 7500 ppm) were considered as treatment-related and adverse.
The significantly reduced body weight gain of male animals in test group 1 (1500 ppm) from study day 7 to 13 and female animals of test group 2 (5000 ppm) from gestation day 10 to 14 were considered as incidental and spontaneous in nature. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- In male animals of test group 3 (15000 ppm) food consumption was decreased significantly from premating day 0 to 7 (-31.9%) and therefore the value of the overall interval for premating (day 0 to 13) was significantly decreased as well (-23.2%).
Food consumption in female animals of test group 3 (15000 / 7500 ppm) was also decreased during premating with a maximum of -23.6% (day 0 to 7) and -18.7% for the overall premating interval (non-statistically significant), and from gestation day 7 to 20 with a maximum of -25.8% (GD 14 to 20) and -16.5% for the overall gestation interval. Furthermore, during the entire lactation period (-28.8% PND 1-13) with a maximum of -32.1% (PND 10 to 13). The statistical analysis was not performed for the test group 3 (7500 ppm) in lactation because of the sample size of n=2. These findings were considered as treatment-related and adverse.
In females of test group 1 (1500 ppm) a slightly increased food consumption was determined during premating (15%, day 0 to 13). This finding was considered as incidental and not treatment related.
In male animals of test groups 1 and 2 (1500 and 5000 ppm) and female animals of test group 2 (5000 ppm) no changes in food consumption were observed at any time point. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, treatment-related
- Description (incidence and severity):
- Water consumption was significantly decreased in males of test group 3 (15000 ppm) over the entire study period with a maximum of -29.3% (day 10 to 13) and in males of test group 2 (5000 ppm) from premating day 10 to 13 (-16.6%). The finding in the test group 3 was assessed as treatment-related and adverse, while the isolate single finding in test group 2 was assessed as incidental and not related to treatment.
Decreased water consumption was also determined in females of test group 3 during the entire premating period with a maximum of -36.5% (premating day 3 to 7), during entire gestation with a maximum of -47.1% (GD 19 to 20) including one non statistically significant interval from gestation day 6 to 7 (-20.0%) and during entire lactation period with a maximum of -47.7% (lactation day 12 to 13; the statistical analysis was not performed for the test group 3 (15000 / 7500 ppm) because of the sample size of n=2 during lactation). An palatability issue of the test item caused by the strong smell and presumably bad taste of the test substance in the drinking water is likely by the test substance being an odorous substance. However, since the no palatability issue was observed in the test group 2 (5000 / 2500 ppm) while during lactation in test group 3 (7500 ppm, aiming a comparable substance intake then before) the decreased water consumption was still observed in the same degree than before (15000 ppm), the decreased water consumptions in test group 3 were considered as test substance-related and adverse.
Intake of test substance
Mean test substance intake (mg/kg bw/d; minimum value/maximum value):
Test group 1 (1500 ppm (750 ppm during lactation))
- F0 males: 94.2 (89.9 / 98.3)
- F0 females premating: 149.8 (146.5 / 153.1)
- F0 females gastation period: 159.0 (141.5 / 173.7)
- F0 females lactation period: 136.1 (100.3 / 164.7)
- F0 females balanced mean of all periods: 149.9
Test group 2 (5000 ppm (2500 ppm during lactation))
- F0 males: 297.3 (283.0 / 308.3)
- F0 females premating: 462.8 (450.5 / 475.0)
- F0 females gastation period: 525.6 (454.9 / 595.4)
- F0 females lactation period: 468.8 (378.8 / 554.0)
- F0 females balanced mean of all periods: 491.8
Test group 3 (15000 ppm (7500 ppm during lactation)):
- F0 males: 769.5 (742.1 / 798.6)
- F0 females premating: 1027.3 (955.3 / 1099.3)
- F0 females gastation period: 1138.8 (966.3 / 1226.4)
- F0 females lactation period: 1016.5 (933.7 / 1114.9)
- F0 females balanced mean of all periods: 1072.7 - 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 males of test group 3 (15000 ppm) hemoglobin values were significantly decreased, but the mean was within the historical control range (males, hemoglobin 8.5-9.7 mmol/L) and therefore, this change was regarded as incidental and not treatment related. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment-related changes among clinical chemistry parameters were observed.
At lactation day 14 in dams of test group 3 (15000 ppm) triglyceride and total bilirubin values were decreased. Statistics could not be performed, because blood samples of only two dams in this test group were measured. Triglyceride values were marginally below, total bilirubin values within the historical control range (dams, triglycerides 1.19-3.20 mmol/L; total bilirubin 1.03-1.81 μmol/L). However, triglycerides were the only altered parameter among these individuals and therefore, this change was regarded as maybe treatment related but nonadverse.
Total bilirubin change was regarded as incidental and not treatment related.
In males of test group 3 (15000 ppm) sodium and chloride values were significantly decreased, but the values were within historical control ranges (males, sodium 140.1-145.8 mmol/L; chloride 95.2-104.8 mmol/L). Therefore, these alterations were regarded as incidental and not treatment related. - Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment related, adverse changes among urinalysis parameters were observed.
In males of test group 3 (15000 ppm), urine volume was decreased, and urine specific gravity was increased. These alterations reflected an adaptive effect towards reduced fluid income into the kidneys. Therefore, these changes without any other findings in the renal tract were regarded as maybe treatment-related, but non-adverse. - Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- Functional observational battery:
Deviations from "zero values" were obtained in quantitative parameters in male and female animals. Without a dose-response relationship or occurred in single animals only, these observations were considered as incidental. Based on the low number of females with litters, the samples size is only two in test group 3 (15000 ppm).
The following examinations were performed during FOB and are assessed individually:
Home cage observations: No test substance-related effects were observed.
Open field observations: No test substance-related effects were observed.
Sensorimotor tests/reflexes: No test substance-related effects were observed.
Quantitative parameters: No test substance-related effects were observed.
Motor activity measurement:
Regarding the overall motor activity and single intervals, no test substance-related deviations were noted for male and female animals of any test group. Based on the low number of females with litters, the samples size is only two in test group 3 (15000 ppm). - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Absolute weights:
- Male animals: terminal body weight: 97% (test group 1), 99% (test group 2), 94%** (test group 3)
- Female animals: spleen: 119%* (test group 1), 115% (test group 2), 103% (test group 3)
*p ≤ 0.05; **p ≤ 0.01
All other mean absolute weight parameters did not show significant differences when compared to the control group 0.
Adrenal glands, brain, heart, kidneys, liver, spleen and thymus of only two test group 3 females were weight, because of these organs, weights of 5 females with litters only has to be recorded according to the study plan. Test group 3 had only two females with litters. Therefore, statistics of the respective organ weights in test group 3 females could not be
performed.
Relative weights:
- Male animals:
kidneys: 105% (test group 1), 106% (test group 2), 119%** (test group 3)
testes: 109%** (test group 1), 99% (test group 2), 109%* (test group 3)
- Female animals:
spleen: 116%* (test group 1), 116% (test group 2), 105% (test group 3)
*p ≤ 0.05; **p ≤ 0.01
All other mean relative weight parameters did not show significant differences when compared to the control group 0.
The significantly decreased terminal body weight in male animals of test group 3 (-6%) lay slightly below the range of historical control values (see PART III, Supplement) and was regarded as treatment related.
The significantly increased relative kidney weights in male animals of test group 3 were assumed to be treatment related. They lay slightly above the range of historical control values.
The significantly increased absolute and relative spleen weights in test group 1 females and the significantly increased relative testes weights in test groups 1 and 3 males were regarded as incidental, because there was neither a dose response relationship nor a correlating histopathological finding. - 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.
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Treatment-related findings were observed in kidneys of test group 3 males and uteri of test group 3 females.
Kidneys of test group 3 males showed a minimal to moderate severity of basophilic tubules, mostly in both kidneys, whereas the control animals only showed a minimal severity of basophilic tubules which occurred in almost all cases unilaterally. The slightly increased severity of basophilic tubules in kidneys of test group 3 males was regarded as treatment related.
The increased storage of pigment in uteri of test group 3 females was regarded as treatment related. The pigment was brown to golden, granular, and was mainly located within the cytoplasm of macrophages. The pigment laden macrophages were loosely arranged within the endometrial stroma, beneath the endometrium and between endometrial glands. The Fe Perls stain (detection of trivalent iron, hemosiderin) and the Fe Turnbull stain (detection of bivalent iron) were positive. Therefore, the pigment was interpreted to be the degradation product of iron-containing molecules, e.g. hemosiderin. The diagnosis does not include pigment that was present in implantation sites.
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. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Description (incidence and severity):
- Thyroid hormones:
In parental males (test groups 1, 2 and 3; 1500, 5000, 15000 ppm), no treatment-related alterations of T4 and TSH levels were observed. - Dose descriptor:
- NOAEL
- Remarks:
- (systemic toxicity)
- Effect level:
- 5 000 ppm
- Based on:
- test mat.
- Remarks:
- corresponding to 770 mg/kg bw/d for males and 492 mg/kg bw/d for females
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical signs
- food consumption and compound intake
- mortality
- water consumption and compound intake
- Critical effects observed:
- no
Reference
The various analyses confirmed
· the stability of the test-substance in drinking water over a period of 7 days was given,
· the solutions were considered to be homogenous without further analysis, as the test substance was completely miscible with drinking water,
· the values of the test substance in drinking water were found to be in a range of 90.1%-98.7% of the nominal concentrations and therefore were within the range of the specification of the test facility (90.0 – 110.0%).
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 492 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
Repeated dose toxicity: inhalation - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: inhalation
- 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 412 (Subacute Inhalation Toxicity: 28-Day Study)
- Deviations:
- yes
- Remarks:
- Exposure period 2 weeks
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Crl:CD(SD)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: approximately 7 weeks old
- Weight at study initiation: male (196 to 233 g), female (154 to 189 g)
- Housing: individually in clean, stainless steel, wire-mesh cages
- Diet: Rodent LabDiet 5002
- Water: reverse osmosis-treated drinking water
- Diet and water restrictions:
during acclimation to the nose-only restraint and the exposure period: no food and water;
prior to the scheduled necropsy, food was offered for at least 2 hours after each animal's final exposure prior to removal for the overnight fasting period, during the period of fasting prior to necropsy: no food, water ad libitum;
- Acclimation period: minimum 13 days; 5 days prior to the initiation of exposure, all animals were acclimated to nose-only exposure tubes; restraint acclimation was performed for approximately 1 hour for the first day, 2 hours for the second day, 3 hours for the third day, 4 hours for the fourth day and 6 hours for the fifth day
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- inhalation: aerosol
- Type of inhalation exposure:
- nose only
- Vehicle:
- air
- Remarks on MMAD:
- MMAD / GSD: Aerosol particle size determinations were conducted for each test substance exposure system using a 7-stage brass cascade impactor. Pre-weighed, glass-fiber filters were used as the collection substrates.
The mean MMAD and GSD ranged between 2.2-2.4 and 2.36-2.54, respectively - Details on inhalation exposure:
- An aerosol atmosphere was generated using a 6-jet Collison nebulizer.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Exposure concentrations of the test material (combined vapor and aerosol) within each nose-only system were determined using an appropriate gas chromatography (GC) method. Concentrations were determined at approximately 60 minute intervals for each test material exposure system and at least once daily for the control system.
- Duration of treatment / exposure:
- 2 weeks
- Frequency of treatment:
- 6 hours per day, 5 days per week
- Remarks:
- Doses / Concentrations:
0.70, 7.0 and 70 mg/m3 (equivalent to 0.1, 1 and 10 ppm)
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
0.84, 6.4 and 73 mg/m3
Basis:
analytical conc. - No. of animals per sex per dose:
- 10
- Control animals:
- other: exposed to filtered air
- Positive control:
- Not applicable
- Observations and examinations performed and frequency:
- MORTALITY AND MORBIDITY: Yes
- Time schedule: twice daily
CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to exposure, at the approximate midpoint of each exposure for visible signs of animals in nose-only exposure restraint tubes and at 0-1 hour following exposure on exposure days and once daily on non-exposure days. Detailed physical examinations were performed during the pretest period, at randomization, weekly during the exposure period and prior to the scheduled necropsy for each animal.
BODY WEIGHT: Yes
- Time schedule for examinations: during the pretest period, at randomization, prior to the first exposure and twice weekly for the duration of the study, including the day before the scheduled necropsy
FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Yes
- Time schedule: during the pretest period and twice weekly during the exposure period
HAEMATOLOGY - COAGULATION PARAMETERS: Yes
- Time schedule for collection of blood: day of necropsy (study day 12/13)
- Animals fasted: Yes (overnight fasting period)
- How many animals: all
Total leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Prothrombin time, Activated partial thromboplastin time, Reticulocyte count, Mean Platelet Volume, Differential leukocyte count, Red cell distribution width , Hemoglobin Distribution Width, Platelet estimate, Red cell morphology
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy (study day 12/13)
- Animals fasted: Yes (overnight fasting period)
- How many animals: all
Albumin, Total protein, Globulin, Albumin/globulin ratio, Total bilirubin, Urea nitrogen, Creatinine, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Glucose, Total cholesterol, Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides, Sorbitol dehydrogenase, Lactate dehydrogenase, Hemolysis, Lipemia, Icterus
SERUM CYTOKINE EVALUATION:
- Time schedule: at necropsy
- How many animals: 5 animals/sex/group (BALF animals)
- samples were taken, but not further assessed for cytokines
BRONCHOALVEOLAR LAVAGE (BAL): Yes
- Time schedule: at necropsy
- How many animals: 5 animals/sex/group (BALF animals)
- Examined parameters: lactate dehydrogenase, total protein, alkaline phosphatase, Total and differential cell counts (Alveolar macrophages, Neutrophils, Lymphocytes, Eosinophils, Basophils), cytokines (TNF-α, IL-5, IL-10, ICAM-1, IFN-γ, IL-4, TGF-β, MCP-1, IL-1β, IL-13, MIP-2, RANTES) - Sacrifice and pathology:
- On the day following the final exposure, all animals were sacrificed and subjected to necropsy. Animals were subjected to either tissue
collection (5 animals/sex/group; histopathology animals) or bronchoalveolar lavage (5 animals/sex/group; BALF animals).
GROSS PATHOLOGY and HISTOPATHOLOGY: Yes
- selected organs were weighed and selected tissues were examined microscopically from 5 animals/sex/group (histopathology animals) at the scheduled necropsy
Organ weights:
Adrenals, Brain, Heart, Kidneys, Liver, Lungs (prior to inflation with fixative), Ovaries with oviducts, Spleen, Testes, Thymus, Thyroid with parathyroids.
Histopathology:
Microscopic examination of the lungs (with bronchi), BALT, nasal tissues, NALT, larynx, trachea, mediastinal and bronchial lymph nodes, and gross lesions were performed for 5 animals/sex/group (Subgroup 1) not assigned for the BAL at the scheduled necropsy. In addition, microscopic examination was performed on the kidneys and liver from 5 animals/sex/group (Subgroup 1) in the control and 70 mg/m3 (high exposure level) groups at the scheduled necropsy. For the nasal cavity and turbinates, 6 sections were examined, including a section containing the nasopharyngeal duct. Three levels of the larynx, including 1 at the base of the epiglottis, were examined. At least 2 sections of the trachea were examined, including 1 longitudinal section through the carina of the extrapulmonary bifurcation of bronchi and 1 transverse section taken at approximately the midpoint of the trachea. - Statistics:
- Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Body weight, body weight change, food consumption, clinical pathology, BALF parameters, and organ weight data were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test
was used to compare the test substance-treated groups to the control group. - Details on results:
- CLINICAL SIGNS AND MORTALITY
All animals survived to scheduled necropsy in all dose goups. Yellow material was found on various body surfaces in the females at dose level of 70 mg/m3. This finding was not considered to be adverse. No other test material related clinical observations at this dose level or dose level of 0.70 mg/m3 and 7.0 mg/m3 were observed.
BODY WEIGHT AND WEIGHT GAIN
Body weights were unaffected by test substance exposure. There were no statistically
significant differences when the control and test substance-exposed groups were
compared.
FOOD CONSUMPTION
Food consumption was unaffected by test substance exposure. There were no statistically
significant differences when the control and test substance-exposed groups were
compared.
HAEMATOLOGY
There were no alterations in hematology parameters that were associated with test substance exposure.
CLINICAL CHEMISTRY
There were no alterations in serum chemistry parameters that were associated with test
substance exposure.
ORGAN WEIGHTS
Organ weights were unaffected by test substance exposure.
GROSS PATHOLOGY
There were no test substance-related macroscopic findings at the scheduled necropsy.
HISTOPATHOLOGY: NON-NEOPLASTIC
There were no test substance-related microscopic findings.
BRONCHOALVEOLAR LAVAGE CLINICAL CHEMISTRY
There were no alterations in BALF chemistry parameters that were associated with test
substance exposure.
BRONCHOALVEOLAR LAVAGE CYTOLOGY
There were no alterations in BALF cytology that were associated with test substance
exposure.
BRONCHOALVEOLAR LAVAGE FLUID CYTOKINE ANALYSIS
There were no alterations in BALF cytokine levels that were associated with test
substance exposure. - Dose descriptor:
- NOAEC
- Effect level:
- 70 mg/m³ air
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: nonadverse clinical observations in the 70 mg/m3 group females
- Critical effects observed:
- not specified
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEC
- 70 mg/m³
- Study duration:
- subacute
- Species:
- rat
Repeated dose toxicity: inhalation - local effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: inhalation
- 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 412 (Subacute Inhalation Toxicity: 28-Day Study)
- Deviations:
- yes
- Remarks:
- Exposure period 2 weeks
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Crl:CD(SD)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: approximately 7 weeks old
- Weight at study initiation: male (196 to 233 g), female (154 to 189 g)
- Housing: individually in clean, stainless steel, wire-mesh cages
- Diet: Rodent LabDiet 5002
- Water: reverse osmosis-treated drinking water
- Diet and water restrictions:
during acclimation to the nose-only restraint and the exposure period: no food and water;
prior to the scheduled necropsy, food was offered for at least 2 hours after each animal's final exposure prior to removal for the overnight fasting period, during the period of fasting prior to necropsy: no food, water ad libitum;
- Acclimation period: minimum 13 days; 5 days prior to the initiation of exposure, all animals were acclimated to nose-only exposure tubes; restraint acclimation was performed for approximately 1 hour for the first day, 2 hours for the second day, 3 hours for the third day, 4 hours for the fourth day and 6 hours for the fifth day
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- inhalation: aerosol
- Type of inhalation exposure:
- nose only
- Vehicle:
- air
- Remarks on MMAD:
- MMAD / GSD: Aerosol particle size determinations were conducted for each test substance exposure system using a 7-stage brass cascade impactor. Pre-weighed, glass-fiber filters were used as the collection substrates.
The mean MMAD and GSD ranged between 2.2-2.4 and 2.36-2.54, respectively - Details on inhalation exposure:
- An aerosol atmosphere was generated using a 6-jet Collison nebulizer.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Exposure concentrations of the test material (combined vapor and aerosol) within each nose-only system were determined using an appropriate gas chromatography (GC) method. Concentrations were determined at approximately 60 minute intervals for each test material exposure system and at least once daily for the control system.
- Duration of treatment / exposure:
- 2 weeks
- Frequency of treatment:
- 6 hours per day, 5 days per week
- Remarks:
- Doses / Concentrations:
0.70, 7.0 and 70 mg/m3 (equivalent to 0.1, 1 and 10 ppm)
Basis:
nominal conc. - Remarks:
- Doses / Concentrations:
0.84, 6.4 and 73 mg/m3
Basis:
analytical conc. - No. of animals per sex per dose:
- 10
- Control animals:
- other: exposed to filtered air
- Positive control:
- Not applicable
- Observations and examinations performed and frequency:
- MORTALITY AND MORBIDITY: Yes
- Time schedule: twice daily
CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to exposure, at the approximate midpoint of each exposure for visible signs of animals in nose-only exposure restraint tubes and at 0-1 hour following exposure on exposure days and once daily on non-exposure days. Detailed physical examinations were performed during the pretest period, at randomization, weekly during the exposure period and prior to the scheduled necropsy for each animal.
BODY WEIGHT: Yes
- Time schedule for examinations: during the pretest period, at randomization, prior to the first exposure and twice weekly for the duration of the study, including the day before the scheduled necropsy
FOOD CONSUMPTION: Yes
- Food consumption for each animal determined: Yes
- Time schedule: during the pretest period and twice weekly during the exposure period
HAEMATOLOGY - COAGULATION PARAMETERS: Yes
- Time schedule for collection of blood: day of necropsy (study day 12/13)
- Animals fasted: Yes (overnight fasting period)
- How many animals: all
Total leukocyte count, Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Platelet count, Prothrombin time, Activated partial thromboplastin time, Reticulocyte count, Mean Platelet Volume, Differential leukocyte count, Red cell distribution width , Hemoglobin Distribution Width, Platelet estimate, Red cell morphology
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: day of necropsy (study day 12/13)
- Animals fasted: Yes (overnight fasting period)
- How many animals: all
Albumin, Total protein, Globulin, Albumin/globulin ratio, Total bilirubin, Urea nitrogen, Creatinine, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Glucose, Total cholesterol, Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides, Sorbitol dehydrogenase, Lactate dehydrogenase, Hemolysis, Lipemia, Icterus
SERUM CYTOKINE EVALUATION:
- Time schedule: at necropsy
- How many animals: 5 animals/sex/group (BALF animals)
- samples were taken, but not further assessed for cytokines
BRONCHOALVEOLAR LAVAGE (BAL): Yes
- Time schedule: at necropsy
- How many animals: 5 animals/sex/group (BALF animals)
- Examined parameters: lactate dehydrogenase, total protein, alkaline phosphatase, Total and differential cell counts (Alveolar macrophages, Neutrophils, Lymphocytes, Eosinophils, Basophils), cytokines (TNF-α, IL-5, IL-10, ICAM-1, IFN-γ, IL-4, TGF-β, MCP-1, IL-1β, IL-13, MIP-2, RANTES) - Sacrifice and pathology:
- On the day following the final exposure, all animals were sacrificed and subjected to necropsy. Animals were subjected to either tissue
collection (5 animals/sex/group; histopathology animals) or bronchoalveolar lavage (5 animals/sex/group; BALF animals).
GROSS PATHOLOGY and HISTOPATHOLOGY: Yes
- selected organs were weighed and selected tissues were examined microscopically from 5 animals/sex/group (histopathology animals) at the scheduled necropsy
Organ weights:
Adrenals, Brain, Heart, Kidneys, Liver, Lungs (prior to inflation with fixative), Ovaries with oviducts, Spleen, Testes, Thymus, Thyroid with parathyroids.
Histopathology:
Microscopic examination of the lungs (with bronchi), BALT, nasal tissues, NALT, larynx, trachea, mediastinal and bronchial lymph nodes, and gross lesions were performed for 5 animals/sex/group (Subgroup 1) not assigned for the BAL at the scheduled necropsy. In addition, microscopic examination was performed on the kidneys and liver from 5 animals/sex/group (Subgroup 1) in the control and 70 mg/m3 (high exposure level) groups at the scheduled necropsy. For the nasal cavity and turbinates, 6 sections were examined, including a section containing the nasopharyngeal duct. Three levels of the larynx, including 1 at the base of the epiglottis, were examined. At least 2 sections of the trachea were examined, including 1 longitudinal section through the carina of the extrapulmonary bifurcation of bronchi and 1 transverse section taken at approximately the midpoint of the trachea. - Statistics:
- Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Body weight, body weight change, food consumption, clinical pathology, BALF parameters, and organ weight data were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test
was used to compare the test substance-treated groups to the control group. - Details on results:
- CLINICAL SIGNS AND MORTALITY
All animals survived to scheduled necropsy in all dose goups. Yellow material was found on various body surfaces in the females at dose level of 70 mg/m3. This finding was not considered to be adverse. No other test material related clinical observations at this dose level or dose level of 0.70 mg/m3 and 7.0 mg/m3 were observed.
BODY WEIGHT AND WEIGHT GAIN
Body weights were unaffected by test substance exposure. There were no statistically
significant differences when the control and test substance-exposed groups were
compared.
FOOD CONSUMPTION
Food consumption was unaffected by test substance exposure. There were no statistically
significant differences when the control and test substance-exposed groups were
compared.
HAEMATOLOGY
There were no alterations in hematology parameters that were associated with test substance exposure.
CLINICAL CHEMISTRY
There were no alterations in serum chemistry parameters that were associated with test
substance exposure.
ORGAN WEIGHTS
Organ weights were unaffected by test substance exposure.
GROSS PATHOLOGY
There were no test substance-related macroscopic findings at the scheduled necropsy.
HISTOPATHOLOGY: NON-NEOPLASTIC
There were no test substance-related microscopic findings.
BRONCHOALVEOLAR LAVAGE CLINICAL CHEMISTRY
There were no alterations in BALF chemistry parameters that were associated with test
substance exposure.
BRONCHOALVEOLAR LAVAGE CYTOLOGY
There were no alterations in BALF cytology that were associated with test substance
exposure.
BRONCHOALVEOLAR LAVAGE FLUID CYTOKINE ANALYSIS
There were no alterations in BALF cytokine levels that were associated with test
substance exposure. - Dose descriptor:
- NOAEC
- Effect level:
- 70 mg/m³ air
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: nonadverse clinical observations in the 70 mg/m3 group females
- Critical effects observed:
- not specified
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEC
- 70 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
Oral exposure
Hydroxycitronellal was administered via the drinking water (ad libitum) to groups of 10 male and 10 female Wistar rats (F0 animals) at concentrations of 0 ppm (test group 0), 1500 ppm (test group 1), 5000 ppm (test group 2) and 15000 ppm (test group 3). Drinking water served as vehicle, control animals were dosed daily with the vehicle only.
The duration of treatment covered a 2-week premating period and mating in both sexes (mating pairs were from the same test group) as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals.
The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. F0 animals were mated for a maximum of two weeks after the beginning of treatment to produce a litter (F1 generation pups). A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, thereafter at weekly intervals. Generally, water consumption of the F0 parents was determined once a week (over a period of 3 or 4 days), as well as in dams during gestation and lactation. Food consumption of the F0 parents was determined regularly once weekly before and after the mating period, as well as in dams during gestation and lactation. In general, the body weights of F0 animals were determined at least twice a week. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and transferred to the Laboratory Pathology for possible further processing. Towards the end of the administration period, a functional observational battery was performed, and motor activity was measured in 5 parental animals per sex and test group. Clinico-chemical and hematological examinations were performed in 5 parental animals per sex and group. Blood samples from all males at termination were taken by puncturing the retrobulbar venous plexus under isoflurane anesthesia. All F0 parental animals were sacrificed by decapitation under isoflurane anesthesia and were assessed by gross pathology. Weights of selected organs were recorded, and a histopathological examination was performed.
Observations and effect on reproductive toxicity of F0 animals and F1 pups are discussed in the corresponding chapter toxicity to reproduction.
The following test substance-related, relevant findings were noted in F0 parental animals:
Test group 3: 15000 ppm (test substance intake in males 770 mg/kg and in females 1073 mg/kg)
Clinical Examinations
- One male (No. 38) with piloerection, closed eyelids, poor general condition and reduced nutritional condition
- One female (No. 138) with closed eyelids, smeared fur at forelimbs, poor general condition and reduced nutritional condition
- One male and one female sacrificed moribund (Nos. 38 and 138) due to poor general condition and reduced nutritional condition on study day 8
- Reduced water consumption in males during entire administration period (maximum -29.3%, study days 10 to 13) and in females during pre-mating (maximum -36.5%, study day 3 to 7), gestation (maximum -47.1%, gestation days 19 to 20) and reduced water consumption during lactation (maximum -47.7%, lactation days 12 to 13)
- Reduced food consumption in males during pre-mating (study days 0 to 13, -23.2%) and in females during gestation (gestation days 0 to 20, -16.5%)
- Reduced food consumption in females during pre-mating (study days 0 to 13, -18.7%, non-statistically significant)) and lactation (lactation days 1 to 13, -28.8%, excluded from analytical statistics because of the sample size of n=2)
- Reduced body weight in male from study day 7 onwards (maximum -7.6%, study day 7) and in females from gestation day 10 onwards (maximum -19.6%, gestation day 20) including lactation with a deviation from control up to -8.1% (excluded from analytical statistics because of the sample size of n=2)
- Significantly reduced body weight gain in males from study day 0 to 13 with body weight loss (-20.6g) from study days 0 to 7 and therefore significantly reduced overall body weight gain (study days 0 to 28)
- Significantly reduced body weight gain in females during pre-mating day 0 to 13 with body weight loss from pre-mating days 0 to 7 (-8.5g), during gestation (from gestation day 10 onwards) and lactation (body weight loss (-3.5g) between lactations days 10 and 13)
Clinical Pathology
- No treatment-related, adverse effects were observed.
Pathology
- Significantly decreased terminal body weight in male animals (-6%)
Test group 2: 5000 ppm (test substance intake in males 297 mg/kg and in females 492 mg/kg) and Test group 1: 1500 ppm (test substance intake in males 94 mg/kg and in females 150 mg/kg)
Clinical Examination, Clinical Pathology and Pathology
- No treatment-related, adverse effects were observed.
Under the conditions of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the oral administration via the drinking water of Hydroxycitronellal to male and female Wistar rats revealed signs of systemic toxicity at a concentration of 15000 ppm.
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 5000 ppm for male for female Wistar rats (770 mg/kg bw/d and 492 mg/kg bw/d, respectively).
In a chronic oral (feeding) repeated dose toxicity study, reported in literature with limited documentation, hydroxycitronellal was administered to rats at concentrations of 0.1% and 0.5% (approx. 50 and 250 mg/kg bw/d) for 2 years (Bär, 1967). The animals were checked in intervals for their appearance, behaviour and body weight. After 2 years the animals were necropsied and liver, kidney, adrenals, heart, spleen, pancreas and brain were examined histopathologically. It has been reported that both dietary levels are well tolerated in rats for the duration of their life-span. However, the available data are from an old feeding study that was only reported in a short summary table and that lacks the rigor, diversity and numbers of animals, multiplicity of dose levels and width of observation of modern studies.
A testing proposal with the registered substance for a Repeated dose 90-day oral toxicity study (test method: OECD TG 408) in rats, oral route has been submitted in order to fullfill standard information requirement as laid down in Annex IX, Section 8.6.2. of the REACH Regulation. An OECD 470 will be implemented in the study design to
Inhalative exposure
In a subacute inhalation study (acc. to OECD TG 412 and GLP), Crl:CD(SD) rats (10 per sex and dose) were treated to aerosolized hydroxycitronellal by nose-only inhalation 6 hours per day on a 5-day per week basis for a period of 2 weeks (minimum of 10 exposures; WIL 2013). Target exposure concentrations were 0.7, 7.0 and 70 mg/m3 (analytical overall mean exposure concentrations: 0.84, 6.4 and 73 mg/m3). Animals were examined for mortality, clinical signs, body weights, food consumption, hematology/ coagulation, clinical chemistry. Bronchoalveolar lavages were performed in 5/10 animals per sex and dose and BALF was assessed for lactate dehydrogenase, total protein, alkaline phosphatase, cytology and cytokines. All animals were sacrificed, subjected to necropsy and selected organs were weighed and examined microscopically in 5/10 animals per sex and dose.
All animals survived to scheduled necropsy. Yellow material was found on various body surfaces in the females at dose level of 70 mg/m3. No other test material related clinical observations at this or lower dose levels were observed. Body weights, food consumption, clinical pathology parameters and organ weights were unaffected by test material exposure and no alterations in BALF clinical chemistry, BALF cytology or BALF cytokine parameters that were associated with test material exposure was found. Furthermore, no test material related macroscopic or microscopic findings were observed in all dose groups.
Overall, hydroxycitronellal-related effects were limited to non-adverse clinical observations in the 70 mg/m3 group females. Therefore, the NOAEC was set at 70 mg/m3 (equivalent to 10 ppm).
Dermal exposure
No key data on repeated dermal exposure of hydroxycitronellal are available.
Justification for classification or non-classification
The present data on repeated dose toxicity do not fulfill the criteria laid down in regulation (EU) 1272/2008, and therefore, a non-classification is warranted. If necessary, the classification will be updated as soon as results from the proposed repeated dose 90-day oral toxicity study in rodents will be available.
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