7-hydroxycitronellal

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 422: rat, drinking water (BASF 2020)

NOAEL (general systemic toxicity) = 5000 ppm for male for female Wistar rats (770 mg/kg bw/d and 492 mg/kg bw/d, respectively)
NOAEL (reproductive performance and fertility) = 5000 ppm for male and female Wistar rats (770 mg/kg bw/d and 492 mg/kg bw/d, respectively)
NOAEL (developmental toxicity) = 5000 ppm (492 mg/kg bw/d)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance 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.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: overnight (from about 16.00 h until 06.30 - 09.00 h of the following morning) for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy (gestation day (GD) 0)
- After successful mating each pregnant female was caged (how): Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.

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 level; 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

Parental animals: Observations and examinations:

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

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.

Oestrous cyclicity (parental animals):

For all females of the pool estrous cycle normality was evaluated before the beginning of the administration period.
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.

Sperm parameters (parental animals):

After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals:
sperm motility, sperm morphology, sperm head count (cauda epididymis), sperm head count (testis)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4 pups/sex/litter as nearly as possible); excess pups were sacrificed under isoflurane anesthesia by decapitation; standardization of litters was not performed in litters with ≤ 8 pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, presence of gross anomalies, clinical observations, postnatal mortality, weight gain, anogenital distance (AGD), presence of nipples/areolae in male pups, blood thyroid hormone concentrations, gross necropsy

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, on study day 31
- Maternal animals: All surviving animals, on study day 62

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following weights were determined in all animals sacrificed on schedule:
Anesthetized animals (final body weight), Epididymides, Ovaries, Prostate (ventral and dorsolateral part together, fixed), Seminal vesicles with coagulating glands (fixed), Testes, Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix
The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):
Adrenal glands (fixed), Brain, Heart, Kidneys, Liver, Spleen, Thymus (fixed)
All paired organs were weighed together (left and right).
- The tissues indicated in Table 4 were prepared for microscopic examination respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 (surplus pups after litter standardization) and 13 (remaining pups after litter standardization) days of age.
- On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution, were transferred to the Pathology Laboratory and were archived without further processing.
- All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically.
- All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

GROSS NECROPSY
- After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.

Statistics:

see table 5

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.
- mating and fertility indices were calculated for F1 litters (for formulas see "Any other information on materials and methods")

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.
- mating, fertility and gestation indices, live birth index, postimplantation loss were calculated for F1 litters (for formulas see "Any other information on materials and methods")

Offspring viability indices:

- viability index, survival index (for formulas see "Any other information on materials and methods")

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. 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.

Absolute weights:
- Male animals: terminal body weight: 97% (test group 1), 99% (test group 2), 94%** (test group 3)
* p ≤ 0.05; ** p ≤ 0.01

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, non-treatment-related

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.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data revealed regular cycles in most of the female animals of all test groups including the control. The mean estrous cycle duration in test groups 0 to 3 was at or between 3.9 to 4.3 days. A treatment-related change regarding the estrous cycle parameters did not occur in any test group.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed.
Regarding spermanalysis in test group 3 (15000 ppm) rat no. 31 has greatly changed parameters. Histopathologically in the epididymis of this male a sperm granuloma was observed. Because this finding was isolated in this test group, it was regarded as spontaneous and not treatment related. As consequence, statistics was performed after exclusion of this outlier.
Sperm motility was significantly decreased in males of test group 2 (5000 ppm). Although the mean motility in this group was below the historical control range, the change was not dose dependent. In test group 3 (15000 ppm) sperm motility was within the historical control range. All other spermanalysis values in the test groups were within historical control ranges (motility 79-93 %, spermatid counts in testis 87-128 Mio/g; sperm counts in cauda epididymidis 501-890 Mio/g; abnormal sperms 5.0-7.5 %). No treatment related findings were observed histopathologically in the male reproductive tract. Therefore, the decreased motility in males of test group 2 was regarded as incidental and not treatment related

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

MALE REPRODUCTION DATA
- Male mating index
No treatment-related changes with regard to the male mating index was observed in any test group.
The male mating indices calculated after the mating period to produce F1 litter were 100% in all test groups.
- Male fertility index
Fertility was proven in all of the F0 parental males of test groups 0 to 2 (0, 1500 and 5000 ppm) within the scheduled mating interval to produce F1 litter. Male animal No. 31 of test group 3 (15000 ppm), which was mated with female No. 131, did not generate F1 pups.
No implants were found at necropsy in female animal No. 131. The male mating partner (animal No. 31) showed macroscopically a focus in one epididymis, that presented as a sperm granuloma histologically. The sperm granuloma was assigned to the infertility of this animal. However, the finding occurred in one single animal and sperm granulomas are common background lesions. Therefore, this finding was regarded to be incidental and not related to the treatment.
Thus, the male fertility index was 100.0% in test groups 0 to 2 (0, 1500 and 5000 ppm) and 88.9% in test group 3 (15000 ppm).
These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data (80-100%).

FEMALE REPRODUCTION DATA
- Female mating index
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until sperm was detected (GD 0) was 2.2 days for test group 0 (0 ppm), 3.6 days for test group 1 (1500 ppm), 2.3 days for test group 2 (5000 ppm) and 2.6 days for test group 3 (15000 ppm).
These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Female fertility index
All sperm positive rats of test groups 0 to 2 (0, 1500 and 5000 ppm) delivered pups. Thus, the female fertility index was 100% in test groups 0 to 2. In test group 3 (15000 ppm), all female animals mated were sperm positive. As female animal No. 131 of test group 3 was sperm positive but had no implants and did not generate pups (this animal did not show relevant gross lesions), the female fertility index was 88.9%.
These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data (80-100%).
- Gestation index
The mean duration of gestation was 22.1 days in test group 0 (0 ppm), 22.3 days in test group 1 (1500 ppm), 22.2 days in test group 2 (5000 ppm) and 22.0 days in test group 3 (15000 ppm).
These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
The gestation index was 100% in test groups 0 to 2 (0, 1500 and 5000 ppm). In test group 3 (15000 ppm) the gestation index was considerably reduced with a value of -25% caused by 6 female animals which had implants but did not generate pups (this animals did not show relevant gross lesions). This finding was considered as treatment related and adverse.
- Live birth indices
The rate of live birth indices were 98.4% in test group 0, 97.5% in test group 1 (1500 ppm), 98.5% in test group 2 (5000 ppm) and 100% in test group 3 (15000 ppm). There were 2 stillborn pups recorded in control group (1.6%), 3 stillborn pups recorded in test group 1 (1500 ppm; 2.5%) and 2 stillborn pups recorded in test group 2 (5000 ppm; 1.5%).
The values of all test groups reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data (pups liveborn 89.3 - 100%, pups stillborn 0.0 - 10.7%).
- Postimplantation loss
The postimplantation loss was 5.5% in test group 0 (0 ppm), 6.7% in test group 1 (1500 ppm) and 5.1% in test group 2 (5000 ppm).
These values were within the normal range of biological variation inherent in the strain of rats used for this study.
In test group 3 (15000 ppm) the postimplantation loss was significantly increased with 75%. This finding was considered as treatment-related and adverse.

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:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

Dose descriptor:

NOAEL

Remarks:

(reproductive performance and fertility)

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:

reproductive performance

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

In each test group including control group some pups died during the lactation period.
All other F1 pups in test groups 0 to 3 did not show clinical signs up to scheduled sacrifice on PND 4 and PND 13.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The pup mortality was based on total number of stillborn pups, dead pups, pups sacrificed moribund and cannibalized pups. The number of pups which died from PND 1 to 4 was increased in test group 3 (7500 ppm) considering the lower number of pups delivered (n=23). This is also shown in the low viability index of 61.2% in this test group. This finding was assessed as treatment related an adverse.
The values for test groups 0, 1 and 2 reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean pup body weights were reduced during the entire lactation pup period in test group 3 (7500 ppm) with a maximum of -14.4% on post-natal day 1 (PND1). The reduction of pup body weights is nearly the same for both sexes. Thereby, no statistical analysis was possible to be performed since only two litters were given. However, the mean pup weights were outside of the historical control range for postnatal day 1 for males, females and the combination of both genders. Mean pup body weights of test group 3 were in the range of historical control data from PND 4 onwards. Mean pup body weight gain was only slightly reduced in test group 3 (7500 ppm) in the overall interval (PND 1 to 13). Reduction of body weight gain started from PND 4 to 7 and continued to PND 13. These findings were considered as treatment-related and adverse.
Mean pup body weights and pup body weight change values were comparable to control in test groups 1 and 2.
Two male runts and three female runts in test group 0 (0 ppm), four male runts and one female runt in test group 1 (750 ppm) and one male runt each in test group 2 and 3 (2500 ppm and 7500 ppm) were found.
A relation to dosing was not observed, test substance-related effects did not occur.

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

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

No test substance-related effects in anogenital distance and index were observed in test groups 1-3 (750, 2500 as well as 7500 ppm, respectively) when compared to the controls. The slightly reduced anogenital distance in male pups of test group 3 (7500 ppm) was well in the range of historical control data.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

In test groups 1 - 3 (750, 2500 and 7500 ppm, respectively), the apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

A few F1 pups showed spontaneous findings at gross necropsy, such as postmortem autolysis, discolored testis, empty stomach, diaphragm hernia and situs inversus. One male pup in test group 1 (750ppm) was partly cannibalized. Three pups were not assessed because those were missing (cannibalized).
In test group 3 (7500 ppm) the number of animals with signs (affected [%]) was increased in male (+26.7%) and female (+50%) pups compared to the other test groups. Most of the findings were related to postmortem assessment, like autolysis or not assessed (in males +20 out of +27% and in females +25 out of +50%). The further finding in females were an empty stomach (+25%), which is given in the historical control data and reached in this study a higher relative value based on the low number of pups delivered in test group 3. Therefore, the necropsy findings of test group 3 does not indicate further morphological manifestation of developmental toxicity. However, these findings linked to reduced viability were considered as treatment-related and adverse.
No treatment-related findings were observed for test group 1 (750 ppm) and 2 (2500 ppm).

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Litter data
- Pup number and status at delivery
The mean number of delivered F1 pups per dam was 12.6 in test group 0 (0 ppm), 12.1 in test group 1 (1500 ppm), 13.0 in test group 2 (5000 ppm) and 11.5 in test group 3 (15000 ppm).
These values were within the normal range of biological variation inherent in the strain of rats used for this study.
- Pup viability index/mortality
The viability index indicating pup mortality between PND 0 and 4 was 98.5% in test group 0 (0 ppm), 98.3% in test group 1 (750 ppm), 100% in test group 2 (2500 ppm) and 61.2% in test group 3 (7500 ppm). Viability index in test group 3 is considerably reduced and therefore assessed as treatment-related and adverse.
The survival index indicating pup mortality between PND 4 and 13 was 100% in test groups 0, 2 and 3 and 98.8% in test group 1.
Concerning viability index the values for test groups 0, 1 and 2 and concerning survival index the values for test groups 0 to 3 reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Sex ratio
In test groups 0, 1 and 2 (0, 750 and 2500 ppm), the sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups. Slight differences were regarded to be spontaneous in nature. In test group 3 (7500 ppm) the sex distribution and sex ratio differed from control group and is slightly out of the range of historical control data (36.6 – 60.5% males and 41.5 – 63.4% females). This slight deviation from the sex ratio in the historical control data was considered mainly by the low number of pups, delivered from 2 females only, in test group 3 in comparison to the other test groups and control of this study (121-130 pups), which reflects the normal expectation in this study type.
Therefore, this finding was considered as incidental and not related to treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

5 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

15 000 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

492 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Hydroxycitronellal was administered via the drinking water to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels 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 administered 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), the entire gestation period (about 22 days) as well as 13 days of lactation period in females up to the day of scheduled sacrifice of the animals.

Regarding clinical examinations, signs of general systemic toxicity were observed in male and female parental animals of test group 3 (15000 ppm). Water consumption, food consumption and body weights were decreased in male and female animals. The body weight at the end of the administration period was -7% in males on study day 28 and -8% in females on lactation day 13. Furthermore, one male and one female of this test group had to be sacrificed moribund after 8 days of repeated administration. However, all other parental animals tolerated the further repeated administration during the study. These changes were assessed to be related to treatment and adverse.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the test substance of 15000 ppm, the highest dose tested.

Regarding pathology, target organs were the kidneys in male animals and the uterus in female animals. The significantly decreased terminal body weight in male animals of test group 3 (15000 ppm: -6%) was regarded as treatment-related and adverse. The significantly increased relative kidney weights in male animals of test group 3 lay slightly above the range of historical control values (see PART III, Supplement). They were assumed to be treatment-related but not adverse, because only the relative weights were significantly increased, and the weight increase was mild. Additionally, kidneys of test group 3 males showed a slightly increased severity of basophilic tubules, which was regarded as treatment related. Because basophilic tubules are a very common background finding and the severity of this finding was only mildly above the severity range that is known from control animals, the increased severity of basophilic tubules in this study was interpreted as not adverse. The increased storage of pigment in uteri of test group 3 females was regarded as treatment related. Based on the staining properties of the pigment it was interpreted to be the degradation product of iron-containing molecules, e.g. hemosiderin. It occurred in females, showing a post implantation loss. Therefore, it was regarded as a secondary and non-adverse change.

Fertility indices for male and female animals were not impaired by test-substance administration up to the limit dose level of 15000 ppm in drinking water. Gestation index was severely reduced and post implantation loss was increased in test group 3 (15000 ppm). Live birth indices of pups in all test groups were not influenced.

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, impairments of reproductive performance and developmental 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).

The NOAEL for reproductive performance and fertility was also set to 5000 ppm for male and female Wistar rats (770 mg/kg bw/d and 492 mg/kg bw/d, respectively).

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

492 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Hydroxycitronellal was administered via the drinking water to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels 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 administered 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), the entire gestation period (about 22 days) as well as 13 days of lactation period in females up to the day of scheduled sacrifice of the animals.

Regarding clinical examinations, signs of general systemic toxicity were observed in male and female parental animals of test group 3 (15000 ppm). Water consumption, food consumption and body weights were decreased in male and female animals. The body weight at the end of the administration period was -7% in males on study day 28 and -8% in females on lactation day 13. Furthermore, one male and one female of this test group had to be sacrificed moribund after 8 days of repeated administration. However, all other parental animals tolerated the further repeated administration during the study. These changes were assessed to be related to treatment and adverse.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the test substance of 15000 ppm, the highest dose tested.

Regarding pathology, target organs were the kidneys in male animals and the uterus in female animals. The significantly decreased terminal body weight in male animals of test group 3 (15000 ppm: -6%) was regarded as treatment-related and adverse. The significantly increased relative kidney weights in male animals of test group 3 lay slightly above the range of historical control values (see PART III, Supplement). They were assumed to be treatment-related but not adverse, because only the relative weights were significantly increased, and the weight increase was mild. Additionally, kidneys of test group 3 males showed a slightly increased severity of basophilic tubules, which was regarded as treatment related. Because basophilic tubules are a very common background finding and the severity of this finding was only mildly above the severity range that is known from control animals, the increased severity of basophilic tubules in this study was interpreted as not adverse. The increased storage of pigment in uteri of test group 3 females was regarded as treatment related. Based on the staining properties of the pigment it was interpreted to be the degradation product of iron-containing molecules, e.g. hemosiderin. It occurred in females, showing a post implantation loss. Therefore, it was regarded as a secondary and non-adverse change.

The viability index as indicator for pup mortality between postnatal day 0 and 4 was reduced in test group 3 (15000 / 7500 ppm). Furthermore, pup weights at a dose level of 7500 ppm were decreased on postnatal day 1. In correlations to that the number of findings in pup necropsy observations were increased. From postnatal day 4 onwards no treatment-related pup mortality was observed anymore, but the deviation from control in body weight did not decrease over time. 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.

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, impairments of reproductive performance and developmental 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).

The NOAEL for developmental toxicity was 5000 ppm (492 mg/kg bw/d).

 

In addition, in order to fulfill information requirements stated in column 1 Annex IX of REACH Regulation for substances manufactured or imported in quantities of 100-1000 tpa, a prenatal developmental toxicity study in a first species (rat) according to OECD 414 is proposed.

Justification for classification or non-classification

Untill further clarification is provided, no classification on fertility or developmental toxicity according to Regulation (EC) No 1272/2008 is proposed.