3,7-dimethyloct-6-enenitrile

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 443 (BASF, 2021):

NOAEL (general, systemic toxicity, F0, males)= 400 mg/kg bw/d

NOAEL (general, systemic toxicity, F0, females) = 100 mg/kg bw/d

NOAEL (general, systemic toxicity, F1, males) = 100 mg/kg bw/d

NOAEL (general, systemic toxicity, F1, females) = 400 mg/kg bw/d

The NOAEL (fertility and reproductive performance, F0) = 400 mg/kg bw/d

NOAEL (developmental toxicity, F1) = 400 mg/kg bw/d

 

OECD 415 (RIFM, 2011):

NOAEL (general, systemic toxicity, F0)= 200 mg/kg bw/d

NOAEL (general, systemic toxicity, F1) = 500 mg/kg bw/d

The NOAEL (fertility and reproductive performance, F0) = 500 mg/kg bw/d

NOAEL (developmental toxicity, F1) = 500 mg/kg bw/d

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at arrival: Males: 37 days, Females: 65 days
- Weight at study assignment: Males: 129-160 g, females: 224-266 g
- Housing: P Generation Rats/F1 Generation Litters: P generation rats were individually housed in stainless steel, wire-bottomed cages, except during the cohabitation and postpartum periods. During cohabitation, each pair of male and female rats was housed in the male rat's cage. Beginning no later than DG 20, P generation female rats were individually housed in nesting boxes until they either naturally delivered litters or were euthanized on DG 25. Each dam and delivered litter was housed in a common nesting box during the postpartum period. F1 Generation Pups/Rats: After weaning, F1 generation pups selected for continued evaluation were individually housed in stainless steel, wire-bottomed cages. Pups selected for continued observation that had a body weight of less than 28 grams were pair housed for one week in a nesting box to optimize the conditions under which pups are transition from group to individual housing. Bed-o’cobs bedding (The Andersons Industrial Products Group, Maumee, OH) was used as the nesting material. Chewable Nylabones were supplied to all rats during the course of the study.
- Diet: Rats were given ad libitum access to Certified Rodent Diet #5002 meal (PMI Nutrition International, Inc., St. Louis, MO) in individual feeders.
- Water: Local water that had been processed by passage through a reverse osmosis membrane (R.O. water) was available to the rats ad libitum from an automatic watering access system and/or individual water bottles attached to the cages. Chlorine was added to the processed water as a bacteriostat.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30-70
- Air changes (per hr): at least ten changes per hour of 100% fresh air that has been passed through 99.97% HEPA filters
- Photoperiod (hrs dark / hrs light): 12-hours light:12-hours dark fluorescent light cycle was maintained. Each dark period began at 1900 hours ± 30 minutes.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

VEHICLE
- Concentration in vehicle: 0, 18.75, 50, 125 mg/mL for dose groups 0, 75, 200, and 500 mg/kg/day respectively
- Amount of vehicle (if gavage): 4 mL/kg

Details on mating procedure:

Following randomization to study groups, consecutive order was used to assign P generation rats to cohabitation, one male rat per female rat within each dosage group. One hundred and ten virgin female rats were cohabitated with 110 male rats, one male rat per female rat, except for male rat 777 (Group IV, 500 mg/kg/day), which was cohabitated with a second female due to the unscheduled euthanasia of male rat 796. The cohabitation period consisted of a maximum of 13 days. Female rats with spermatozoa observed in a smear of the vaginal contents and/or a copulatory plug observed in situ were considered to be at DG 0 and assigned to individual housing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Concentration and homogeneity of the prepared formulations were verified during the course of this study. Quadruplicate samples were taken on the first day of preparation and from each concentration on the last day of preparation on which all dose formulation concentrations were prepared.
- Stability was determined for the highest concentration as soon as possible on the first day prepared and after room temperature, protected from light storage for 17 days. For each set of stability conditions and timepoints listed, a quadruplicate set of samples was taken from the highest concentration prepared. At the scheduled timepoints after the initial concentration analysis, a duplicate set of samples was analyzed.
- Results for all dose formulations met the acceptance criteria for concentration (within ±15% of nominal concentration) and homogeneity (≤5% relative standard deviation) with the exception of the end of study samples for Group II (18.75 mg/mL), Group III (50 mg/mL), Group IV (125 mg/mL) which were out of specification (+27.3%, +20.3% and +20.0%, respectively). Time zero stability samples at 125 mg/mL were not within the acceptable limits of ±15% of nominal concentration, (+25.6%). However, the result was accepted and used as the reference concentration for formulation stability evaluations.
-Dose formulations at a concentration of 125 mg/mL were considered stable at 22°C ± 5°C when protected from light for 17 days. Stability of citronellyl nitrile formulations at concentrations of 2.5 mg/mL and 75 mg/mL was determined, and the prepared formulations at these concentrations were stable for 21 days when stored at 22°C ± 5°C, protected from light. Based on review of the dose formulation preparation data as well as the analytical method, there were no apparent causes for the out of specification concentration results that occurred at the end of this study. However, these out of specification concentration results did not affect the scientific interpretation of the study results.

Duration of treatment / exposure:

- P Generation Rats: P generation male rats were given the test substance and/or the vehicle once daily beginning 84 days before cohabitation, through cohabitation (maximum of 13 days), and continuing until the day before scheduled euthanasia for a total of 126 to 129 dosages. P generation female rats were given the test substance and/or the vehicle once daily beginning two weeks before cohabitation, through cohabitation (maximum of 13 days), and continuing through DG 25 (rats that did not deliver) or day 22 postpartum (rats that delivered a litter).
- F1 Generation Pups: F1 generation pups were not directly administered the test substance and/or the vehicle, but may have been possibly exposed to the test substance and/or the vehicle during maternal gestation (in utero exposure) or via maternal milk during the lactation period.

Frequency of treatment:

daily, no dam missed more than one daily dosage administration.

Details on study schedule:

- Surviving male rats were euthanized after completion of the cohabitation period. Female rats that delivered a litter and pups not selected for continued evaluation were euthanized on day 22 of lactation (DL 22). Female rats that did not deliver a litter were euthanized on day 25 of presumed gestation (DG 25). F1 generation rats selected for continued evaluation were euthanized on day 60 (± 3) postpartum.

Remarks:

Doses / Concentrations:
75, 200, 500 mg/kg/day
Basis:
nominal in diet

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosages were selected on the basis of a dosage-range reproduction study. Eight rats per sex per group were dosed via gavage with 0 (Vehicle), 10, 30, 100, or 300 mg/kg/day citronellyl nitrile in a corn oil vehicle. Dosing occurred 14 days prior to cohabitation, through cohabitation, gestation, and postpartum day 5 or the day of euthanasia. Absolute and relative feed consumption values were reduced in males from the 300 mg/kg/day dosage group. There were no clinical observations, body weight, body weight gain, mating or fertility parameters, or organ weight changes considered related to treatment in the male rats. In the female rats there were no clinical observations, body weight, body weight gain, feed consumption, estrous cycling, mating or fertility parameters, or natural delivery observations considered related to treatment. Based on these data, a higher dose of 500 mg/kg/day was selected to ensure that sufficient toxicity was observed.

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS: Yes
- Rats were observed for viability twice each day of the study and for clinical observations and general appearance at least once weekly during the acclimation period. Observations for clinical signs, abortions, premature deliveries and deaths were made daily before dosage administration, between one and two hours following dosage administration and at the end of the normal working day and on the day euthanasia occurred. These observations were recorded more frequently than cited above on occasion during this study.

BODY WEIGHT: Yes
- Body weights for the male and female rats were recorded at least weekly during the acclimation period, on the first day of dosage administration, weekly during the dosage period and once on the day of euthanasia. Body weights were also recorded for the female rats on DGs 0, 7, 10, 14, 18, 21 and 25 (for rats that did not deliver a litter) and on DLs 1, 5, 8, 11, 15 and 22 (terminal body weight).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Feed consumption values for the male and female rats were recorded weekly during the acclimation and dosage periods, except during cohabitation. Feed consumption values were also recorded for female rats on DGs 0, 7, 10, 14, 18, 21 and 25 (for rats that did not deliver a litter) and on DLs 1, 5, 8, 11 and 15. Because pups begin to consume maternal feed on or about DL 15, feed consumption values were not tabulated after DL 15.

OTHER:
- Female rats were evaluated for adverse clinical signs observed during parturition, duration of gestation (DG 0 to the day the first pup was observed), litter size (all pups delivered) and pup viability at birth. Maternal behavior was evaluated on DLs 1, 5, 8, 15 and 22.

Oestrous cyclicity (parental animals):

Estrous cycling was evaluated daily by examination of vaginal cytology beginning 14 days before scheduled cohabitation and continuing through cohabitation until mating was confirmed.

Sperm parameters (parental animals):

To assess the potential toxicity of the test substance on the male reproductive system, organ weights, sperm evaluation and histopathology were evaluated. The following organs were individually weighed: right testis, left testis, left epididymis (whole and cauda), right epididymis, seminal vesicles (with and without fluid) and prostate. Sperm concentration and motility were evaluated using computer-assisted sperm analysis (CASA). Motility was evaluated by the Hamilton Thorne IVOS by collection of a sample from the left and right vas deferens. A homogenate was prepared from the left cauda epididymis for evaluation by the Hamilton Thorne IVOS to determine sperm concentration/density (sperm per gram of tissue weight). The remaining portion of the left epididymis (corpus and caput), as well as the right epididymis, prostate and seminal vesicles were fixed in 10% NBF for microscopic evaluation. The testes were fixed in Bouin's solution for 48 to 96 hours and then retained in 10% NBF for microscopic evaluation. Additional tissues were weighed and retained, and microscopic evaluations were conducted; all other tissues were discarded.

Litter observations:

- F1 Generation Pups: DL 1 was defined as the day of birth and was also the first day on which all pups in a litter were individually weighed (pup body weights were recorded after all pups in a litter were delivered and groomed by the dam). Litters were examined after delivery to identify the number and sex of pups, stillbirths, live births and gross alterations. Each litter was evaluated for viability twice daily. The pups in each litter were counted once daily. Clinical observations were recorded once daily during the preweaning period. Pup body weights were recorded on days 1 (birth), 5, 8, 11, 15 and 22 postpartum. Anogenital distance was measured for all live F1 generation pups on days 1 and 22 postpartum. Nipple eruption was evaluated for all live F1 generation pups once on day 12 postpartum.
- F1 Generation Rats: Rats were observed for viability twice daily and for clinical observations once daily during the postweaning period. Body weights were recorded weekly during the postweaning period and on the day euthanasia occurred. Feed consumption values were recorded weekly during the postweaning period (only for individually housed rats). Female rats were evaluated for the age of vaginal patency, beginning on day 28 postpartum and continuing until achievement of this developmental parameter or day 40 postpartum. Male rats were evaluated for the age of preputial separation, beginning on day 39 postpartum until achievement of this developmental parameter or day 50 postpartum.

Postmortem examinations (parental animals):

Gross necropsy included an initial physical examination of external surfaces and all orifices, as well as an internal examination of tissues and organs in situ. The following were examined: external and internal portions of all hollow organs; the external surfaces of the brain and spinal column; the nasal cavity and neck with associated organs and tissues; the thoracic, abdominal and pelvic cavities with associated organs and tissues; and the musculo/skeletal carcass. The lungs were perfused with 10% NBF. Adult P generation rats were necropsied and examined for gross lesions. Gross lesions were retained in 10% NBF and shipped to the Principal Investigator for histological examination.
- Male Rats: After completion of the cohabitation period, all surviving male rats were euthanized by carbon dioxide asphyxiation and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed.
- Female Rats: After completion of the 22-day postpartum period, all surviving female rats were euthanized by carbon dioxide asphyxiation and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. The number and distribution of implantation sites was recorded. The ovaries and uterus with cervix were weighed. Rats that did not deliver a litter were euthanized by carbon dioxide asphyxiation on DG 25 and examined for gross lesions. Uteri were examined while being pressed between glass plates to confirm the absence of implantation sites. Uteri of apparently nonpregnant rats were retained in 10% NBF for possible future evaluation. Additional tissues were weighed and retained, and microscopic evaluations were conducted; all other tissues were discarded.
- Early Deaths: Male rats that died or were euthanized before scheduled termination were examined for the cause of death or condition on the day the observation was made. The rats were examined for gross lesions. Tissues were weighed and retained, and microscopic evaluations were conducted. The lungs, trachea and esophagus were perfused and saved in 10% NBF for microscopic evaluation. The heart, liver, kidneys, stomach and spleen were also retained in 10% NBF and evaluated.

Histopathology: Tissues to be examined histologically were routinely processed, embedded in paraffin, sectioned at 5 microns and stained with hematoxylin and eosin. Microscopic examination was performed on all control and high test substance dosage group P generation rats. All gross lesions were examined microscopically.
- The following tissues were weighed and retained in 10% NBF (unless otherwise noted), and microscopic evaluations were conducted: pituitary gland, brain, liver, kidneys, spleen, adrenal glands, gross lesions (including tissue masses), ovaries, mammary gland, vagina, uterus with cervix, testes (Testes fixed in Bouin’s solution for 48 to 96 hours before being retained in neutral buffered 10% formalin), epididymides, seminal vesicles with coagulating gland (with and without fluid), and prostate. All other tissues were discarded.
- Both ovaries were processed for quantitative evaluation of primordial follicles as follows: Each ovary was embedded in separate paraffin blocks. Consistency in orientation of each ovary was imperative, such that one block number was designated for ovary 1 the left ovary, and the other designated for ovary 2 the right ovary. Beginning at 200 microns within the ovary, five sections were taken 100 microns apart. All five sections were mounted on one slide beginning at the labeled end of the slide. The slide was stained with hematoxylin and eosin (H&E). Each section was quantitatively evaluated for primordial follicles, to include small growing follicles, and each animal was evaluated for presence or absence of corpora lutea of lactation. Follicle counts were reported per ovary, and as a total (both ovaries combined). After the five sections were taken from each ovary for follicular enumeration, a section of each ovary was taken and processed for histopathology.
- The postlactional ovary containing primordial and growing follicles as well as the large corpora lutea of lactation were retained. Histopathological examination was performed to detect qualitative depletion of the primordial follicle population. A quantitative evaluation of primordial follicles was conducted for P generation female rats; the number of rats, ovarian section selection and section sample size should be statistically appropriate for the evaluation procedure used. Examination included enumeration of the number of primordial follicles, which were combined with small growing follicles, for comparison of ovaries of rats assigned to treated and control groups.

Postmortem examinations (offspring):

- F1 Generation Pups: Pups were euthanized by an intraperitoneal injection of sodium pentobarbital (pups ≤ 14 days of age). Pups that died before examination of the litter for pup viability were evaluated for vital status at birth. The lungs were removed and immersed in water. Pups with lungs that sunk were identified as stillborn; pups with lungs that floated were identified as liveborn and to have died shortly after birth. Pups with gross lesions were preserved in Bouin's solution for possible future evaluation. Pups that died or were euthanized before scheduled termination were examined for gross lesions and the cause of death or condition on the day the observation was made. Pups found on days 2 to 5 postpartum were preserved in Bouin's solution for possible future evaluation; pups found on days 6 to 22 postpartum were preserved in 10% NBF. All pups not selected for continued evaluation were euthanized by carbon dioxide asphyxiation on day 22 postpartum and examined for gross lesions; gross lesions were preserved in 10% NBF for possible future histopathological evaluation. Necropsy included a single cross-section of the head at the level of the frontal-parietal suture and examination of the cross-sectioned brain for apparent hydrocephaly. Carcasses of pups not selected for continued observation were discarded without further evaluation.
- Male and female rats were euthanized by carbon dioxide asphyxiation on day 60 (± 3) postpartum. A gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Gross lesions were preserved in neutral buffered 10% formalin. The same tissues were weighed, retained, and evaluated as for the parental animals. In addition, the urinary bladder was inflated with neutral buffered 10% formalin for all postweaning F1 generation rats. Following tissue collection, carcasses were discarded without further evaluation.

Statistics:

Adult data were evaluated with the individual rat as the unit measured. Litter values were used in evaluation of pup data, as appropriate. Variables with interval or ratio scales of measurement, such as body weights, feed consumption values and percent mortality per litter were analyzed as described under the
Parametric heading.
- Parametric: Bartlett’s Test of Homogeneity of Variances was used to estimate the probability that the dosage groups have different variances. A nonsignificant result (p> 0.001) indicated that an assumption of homogeneity of variance was not inappropriate, and the data were compared using the Analysis of Variance. If that test was significant (p≤ 0.05), the groups given the test substance were compared with the control group using Dunnett’s Test. If Bartlett’s Test was significant (p≤ 0.001), the Analysis of Variance Test was not appropriate, and the data were analyzed as described under the Nonparametric heading.
- Nonparametric: when 75% or fewer of the scores in all the groups were tied, the Kruskal-Wallis Test was used to analyze the data, and in the event of a significant result (p≤ 0.05), Dunn’s Test was used to compare the groups given the test substance with the control group. When more than 75% of the scores in any dosage group were tied, Fisher’s Exact Test was used to compare the proportion of ties in the dosage group.
Variables that had graded our count scores, such as litter size or the day a developmental landmark appeared, were analyzed using the procedures described under the Nonparametric heading of the schematic.
Clinical observations and other proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution.

MALE

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- There were no treatment-related deaths at any dosage level tested. One P generation male rat in each of the vehicle control, 200 and 500 mg/kg/day dosage group was humanely euthanized on DS 73, 86 or 121 as a result of a suspected gavage accident or a broken palate. In addition, one male rat in the 200 mg/kg/day dosage group was found dead approximately one hour after dosage administration on DS 116. The cause of death f was not determined based on the in-life, postmortem and microscopic observations.
- Clinical signs in P generation male rats that were attributed to treatment with citronellyl nitrile included slight, moderate and extreme excess salivation at 500 mg/kg/day. Slight excess salivation was observed as early as DS 3 and persisted until scheduled euthanasia. Each of these clinical observations occurred in significantly more (p≤0.01) P generation male rats in comparison to the vehicle control group values. All P generation male rats in the 500 mg/kg/day dosage group had slight excess salivation on one or more occasions, 20 of 25 P generation male rats in this same dosage group also had moderate excess salivation, and 4 P generation male rats in the same dose group also had extreme excess salivation. In addition, a low incidence of ungroomed coat (N=4; p≤0.01) occurred at 500 mg/kg/day.
- All other clinical observations were considered unrelated to treatment with citronellyl nitrile.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Body weight gains for P generation male rats were transiently, but significantly reduced (p≤0.05) in the 200 and 500 mg/kg/day dosage groups during the first week of the dosage period (DSs 1 to 8), in comparison to the vehicle control group value. Body weight gains in the 75, 200 and 500 mg/kg/day dosage groups were 95%, 91% and 93% of the vehicle control group value, respectively, on DSs 1 to 8. These reductions were not dosage dependent. Thereafter, body weight gains at 200 mg/kg/day were generally comparable to the vehicle control group values during the remainder of the dosage period. At 500 mg/kg/day, non-significant reductions in body weight gains occurred on DSs 15 to 22 (85% of controls), DSs 36 to 43 (86% of controls), DSs 78 to 84 (77% of controls), DSs 99 to 106 (68% of controls), DSs 106 to 113 (81% of controls) and DSs 113 to 120 (84% of controls). In addition, body weight gains were significantly reduced (p≤0.01; 65% of controls) in this same dosage group on DSs 120 to 127, in comparison to the vehicle control group value. These sporadic reductions prior to cohabitation, and persistent reductions after cohabitation likely contributed to the overall significant reduction (p≤0.01; 90% of controls) in body weight gains that occurred at 500 mg/kg/day for the cumulative dosage period (DSs 1 to 127). Body weights and body weight gains were unaffected by dosages of citronellyl nitrile as high as 200 mg/kg/day. Body weights on DS 127 (the last day of treatment for all male rats prior to euthanasia) were 94%, 97% and 94% of the vehicle control group value in the 75, 200 and 500 mg/kg/day dosage groups, respectively. The statistically significant increase (p≤0.05) in body weight gains that occurred in P generation male rats in the 500 mg/kg/day dosage group on DSs 57 to 64 was considered unrelated to treatment with citronellyl nitrile because the increase was transient and did not affect the overall body weight gain.
- Absolute and relative feed consumption values were unaffected by dosages of citronellyl nitrile as high as 500 mg/kg/day. All values were comparable among the four dosage groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- All sperm parameters evaluated were unaffected by dosages of citronellyl nitrile as high as 500 mg/kg/day.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- All mating and fertility parameters were unaffected by dosages of citronellyl nitrile as high as 500 mg/kg/day. All values were comparable among the four dosage groups and did not significantly differ from the vehicle control group values.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Organs with statistically significant potentially citronellyl nitrile-related weight changes included the brain, liver, kidneys and spleen.
- The absolute and relative (% terminal body weight) weights of the brain were significantly increased (p≤0.01; 4% and 11% of controls, respectively) in the 500 mg/kg/day dosage group, in comparison to the vehicle control group values. In addition, the absolute and relative (% terminal body weight) weights of the liver (35% and 44% of controls, respectively) and spleen (17% and 25% of controls, respectively) were significantly increased (p≤0.01) at 500 mg/kg/day, in comparison to the vehicle control group values. The absolute and relative weights of the left and right kidney were increased or significantly increased (p≤0.05 or p≤0.01; 5% to 14% of controls) at 500 mg/kg/day, as compared to the vehicle control group. There were no microscopic changes in the P generation male rats that could be correlated with the differences in brain, liver, kidney or splenic weights.

GROSS PATHOLOGY AND HISTOPATHOLOGY (PARENTAL ANIMALS)
- There were no test substance-related necropsy observations.
- Terminal body weights for P generation male rats treated with 500 mg/kg/day citronellyl nitrile were slightly reduced (by 6%), in comparison to the vehicle control group value. This reduction did not reach statistical significance, but reflected an overall reduction (significant at p≤0.01) in body weight gains that occurred at 500 mg/kg/day for the cumulative dosage period (DSs 1 to 127). There were no microscopic changes in the P generation male rats.

FEMALE

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- Similar to male rats, the number of P generation female rats with excess salivation (slight and/or moderate) was significantly increased (p≤0.01) in the 500 mg/kg/day dosage group, in comparison to the vehicle control group value. These increases were observed during the precohabitation, gestation and lactation periods. Slight excess salivation was observed as early as DS 10 and persisted into the lactation period, while the first occurrence of moderate excess salivation was on DS 1. All other clinical observations during the precohabitation, gestation and lactation periods were considered unrelated to citronellyl nitrile.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Body weights and body weight gains during the precohabitation, gestation and lactation periods were unaffected by dosages of citronellyl nitrile as high as 500 mg/kg/day. All values were comparable among the four dosage groups.
- Absolute and relative feed consumption values during the precohabitation, gestation and lactation periods were unaffected by dosages of citronellyl nitrile as high as 500 mg/kg/day. All values were comparable among the four dosage groups.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- The number of estrous stages per 14 days was comparable among the four dosage groups during the precohabitation period.
- All mating and fertility parameters were unaffected by dosages of citronellyl nitrile as high as 500 mg/kg/day. All values were comparable among the four dosage groups and did not significantly differ.
- Pregnancy occurred in 24, 22, 25 and 23 of the 25 mated P generation female rats in the 0 (Vehicle), 75, 200 and 500 mg/kg/day dosage groups, respectively. All pregnant dams delivered litters. Natural delivery and litter observations were unaffected by dosages of citronellyl nitrile as high as 500 mg/kg/day.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- Organs with statistically significant potentially citronellyl nitrile-related weight changes included the liver and kidneys.
- Similar to P generation male rats, the absolute and relative (% terminal body weight) weights of the liver were significantly increased (p≤0.01; 15% and 9% of controls, respectively) in the 500 mg/kg/day dosage group, in comparison to the vehicle control group values. In addition, the absolute weights of the left and right kidney were significantly increased (p≤0.01) at 500 mg/kg/day, as compared to the vehicle control group. There were no corresponding change in the ratio of the kidney weights to terminal body weight; however, similar observations occurred in P generation male rats in this same dosage group. In addition, there were no microscopic changes in the P generation female rats that could be correlated with the differences in liver or kidney weights.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- There were no test substance-related necropsy observations. All gross lesions were considered unrelated to treatment with citronellyl nitrile.
- Terminal body weights for P generation female rats treated with 500 mg/kg/day citronellyl nitrile were significantly increased (5%, p≤0.01), in comparison to the vehicle control group value.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- There were no gross or microscopic test substance-related pathology findings observed in the rats evaluated. Microscopic examination of the ovaries in the 500 mg/kg/day group, as compared to the vehicle control group, revealed that the ovaries of all female rats evaluated appeared to be functional with the presence of corpora lutea and growing follicles. A slight increase in the mean number of follicles per animal, along with higher variability, occurred at 500 mg/kg/day compared to the female rats given the vehicle. Two rats in the 500 mg/kg/day dosage group showed comparatively high numbers of primordial follicles most likely influencing both the noted increase from the vehicle control group rats, and the variability as well.

OVARIAN FOLLICLE COUNTS
see above.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

other: Reproductive NOAEL

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no apparent effects on estrous cycling, mating and fertility, reproductive organ weights or natural delivery parameters.

PREWEANING:

CLINICAL SIGNS (OFFSPRING)
- There were no treatment-related clinical signs observed in the F1 generation pups following treatment of P generation rats with citronellyl nitrile at dosages as high as 500 mg/kg/day.

ANOGENITAL DISTANCE MEASUREMENTS AND NIPPLE ERUPTION
- Anogenital distance on days 1 or 22 postpartum in F1 male and female pups was not affected by treatment of P generation rats with citronellyl nitrile at any dosage level tested.
- Nipple eruption did not occur in any male pup at any dosage level tested. All female pups had nipples present on day 12 postpartum, with the exception of two from litter 16228 (F1 generation; 75 mg/kg/day dosage group).

NECROPSY
- There were no gross lesions observed in the F1 generation pups that survived to scheduled necropsy on day 22 postpartum.

POSTWEANING

MORTALITY AND CLINICAL SIGNS (OFFSPRING)
- All F1 generation rats selected for continued evaluation postweaning survived until scheduled euthanasia.
- All clinical observations that occurred in male or female rats during the postweaning period were considered unrelated to treatment of P generation rats with citronellyl nitrile.

BODY WEIGHT (OFFSPRING)
- Body weights and body weight gains of the F1 generation male and female rats during the postweaning period were unaffected by treatment of P generation rats with citronellyl nitrile at dosages as high as 500 mg/kg/day.

FEED CONSUMPTION
- Absolute and relative feed consumption values for F1 generation male and female rats during the postweaning period were unaffected by treatment of P generation rats with citronellyl nitrile at dosages as high as 500 mg/kg/day.

SEXUAL MATURATION (OFFSPRING)
- Sexual maturation in F1 rats was unaffected by treatment of P generation rats with citronellyl nitrile at dosages as high as 500 mg/kg/day.

NECROPSY OBSERVATIONS
- There were no test substance-related necropsy observations.

ORGAN WEIGHTS (OFFSPRING)
- There were no apparent effects of treatment of P generation rats with citronellyl nitrile on the reproductive and nonreproductive tissue weights of the F1 male or female rats at any dosage level tested.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no apparent effects growth and development (including anogenital distance, nipple eruption or sexual maturation) in the F1 generation rats at the highest dosage level tested (500 mg/kg/day).

Reproductive effects observed:

not specified

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

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 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

25 June 2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS

- Premating exposure duration for parental (P0) animals: 10 weeks; according to ECHA Compliance Check Decision CCH-D-2114448631-50-01/F
- Basis for dose level selection:
In a previous OECD No. 414 study (BASF, project No. 30R0333/03R011, 2016), Citronellylnitril was administered as an oily solution to groups of 25 time-mated female rats by gavage at doses of 50, 150 and 450 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. Maternal toxicity was observed at the highest dose level of 450 mg/kg bw/d, namely increased absolute and relative liver weights (19 and 21% above control) and corresponding clinical-pathological changes (increased triglyceride and cholesterol values). Therefore, the no observed adverse effect level (NOAEL) for maternal toxicity was set to 150 mg/kg bw/d.
In a previous OECD No. 408 study (SPL, project No. 2153-0004, 2008), Citronellylnitril was administered by oral gavage at dose levels of 300,100, 30, 10 and 0 mg/kg bw/d for a period of ninety consecutive days. No adverse effects were observed in clinical examinations. In (histo)pathology, centrilobular, hepatocyte enlargement and increased absolute and relative liver weights at 300 (and partly at 100) mg/kg bw/d were observed. These changes were discussed as adaptive and the NOAEL was set to 300 mg/kg bw/d.
In an OECD No. 415 one-generation reproduction study in rats with an evaluation through sexual maturity in the F1 Generation (CLR, study No. TIF00044, 2011), Citronellylnitril was administered by oral gavage to 25 rats per sex and group at dose levels of 500, 200, 75 and 0 mg/kg bw/d. The purpose of this study was to provide information concerning the effects of Citronellylnitrile on gonadal function, estrous cycles, mating behavior, conception, parturition, lactation and the growth and development of offspring up to day 60 postpartum.
In this study, the no-observable-adverse-effect-level (NOAEL) for systemic toxicity was 200 mg/kg/day based on the below mentioned effects. At 500 mg/kg/day, reductions in body weight gains occurred in F0 male rats prior to cohabitation, followed by persistent reductions in weight gain through the end of the study (10% below control). In pathology, increased absolute and relative brain (males: 4 and 11% above control), spleen (males: 17 and 25% above control, respectively), liver (males: 35 and 44%; females: 15 and 9% above control) and kidney weights (males: 5 and 14% above control) occurred in F0 animals at 500 mg/kg/d. Microscopic findings that could be correlated with the changes in organ weights were not observed. The NOAELs for reproductive performance in the F0 and for viability and growth of the F1 generation offspring was greater than 500 mg/kg/d.
Based on the above-mentioned study results, the following dose levels were selected: 25 mg/kg bw/d as low dose, 100 mg/kg bw/d as intermediate dose, 400 mg/kg bw/d as high dose.
- Exclusion of extension of Cohort 1B: according to ECHA Compliance Check Decision CCH-D-2114448631-50-01/F
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B: according to ECHA Compliance Check Decision CCH-D-2114448631-50-01/F
- Exclusion of developmental immunotoxicity Cohort 3: according to ECHA Compliance Check Decision CCH-D-2114448631-50-01/F
- Route of administration: oral (gavage): according to ECHA Compliance Check Decision CCH-D-2114448631-50-01/F
- Choice of species, strain: The rat is the preferred animal species for reproduction studies according to test guidelines. This strain was selected since extensive historical control data were available for Wistar rats.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch number of test material: BASF SE, 19000329U0
- Purity: 95.6 area-%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability of the test material during storage: Expiry date: 01 May 2021
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Homogeneity of the test material: given

OTHER
- Chemical name: 3,7-dimethyloct-6-enenitrile
- Physical state/appearance: Liquid/colorless, clear

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at start of the administration period: 35 ± 1 days
- Weight at study initiation: weight variation did not exceed 20 percent of the mean weight of each sex (P)
- Fasting period before study: no
- Housing: together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Hohenpeißenberg, Germany with the following exceptions:
• During overnight matings (male and female mating partners were housed together), gestation, lactation and females after weaning the animals were housed individually in Polycarbonate cages type III (supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany).
• Dams and their litters were housed together until PND 22 in Polycarbonate cages type III.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: about 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 05 Nov 2019 (TS administration) To: 08 May 2020 (sacrifice of F1 cohort 1B animals)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The test substance preparations were prepared in intervals, which took into account the analytical results of the stability verification.
- For the preparation of the administration suspensions the test substance was weighed in a graduated flask depending on the dose group, topped up with corn oil and intensely mixed with a magnetic stirrer until it was completely homogenized.
- Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0.63, 2.5, 10.0 g/100 mL in low, mid and high dose, respectively
- Amount of vehicle (if gavage): 4 mL/kg bw/d

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: max. 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in corn at room temperature over a period of 7 days had been verified prior to the start of the study.
The applicable form of the test-substance preparation was completely miscible with corn oil and thus a solution. Therefore, the test-substance preparation was considered to be homogenous.
Samples of the test substance preparations were sent to the analytical laboratory during the study period (at the beginning, towards the middle and towards the end) for verification of the concentrations.

Duration of treatment / exposure:

F0 males: 10 weeks (premating) + 2 weeks (mating) + max. 6 weeks (post-mating)
F0 females: 10 weeks (premating) + 2 weeks (mating) + approx. 6 weeks (pregnancy + lactation)
F1 animals (Cohort 1A and 1B): post weaning until an approx. age of 13 weeks

Frequency of treatment:

once daily

Details on study schedule:

F0 generation animals and their progeny:
The male and female animals were about 4 weeks old when they arrived from the breeder. During an acclimatization period of about 7 days, animals with lowest and highest body weights were eliminated from the study and used for other purposes. The 100 male and 100 female animals required for the study were about 5 weeks old at the beginning of treatment and their weight variation did not exceed 20 percent of the mean weight of each sex.
The assignment of the animals to the different test groups was carried out using a randomization program, according to their weight one day before the beginning of the administration period (day -1).
After the acclimatization period, the test substance was administered to the animals orally by gavage, once daily at approximately the same time in the morning. Females in labor were not treated. The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated with the vehicle (corn oil), in the same way. The volume administered each day was 4 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
After a minimum of 10 weeks after the beginning of treatment, males and females from the same dose group were mated, overnight at a ratio of 1 : 1 (for details see: Pairing of F0 generation parental animals).
The females were allowed to deliver and rear their pups (F1 generation pups) until PND 4 (standardization) or PND 21 or 22 (depending on the cohort). Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts which were subjected to specific postweaning examinations.
On PND 4 blood samples were collected from 10 surplus (culled) F1 pups per sex and group. On PND 22 blood samples were collected from 10 surplus F1 pups per sex and group.
Blood samples were taken from 10 animals per test group of the F0 parental animals and cohort 1A animals.
Before weaning of the F1 pups the F0 generation parental male animals were sacrificed.
After weaning of F1 pups the F0 generation parental female animals were sacrificed.

F1 (rearing animals):
Before weaning of the F1 generation pups on PND 21, 45 male and 45 females per group were randomly selected (selection see below), to be placed into cohorts according to the scheme presented under 3.7.3. Obvious runts (those pups whose body weight was - 25% below the mean body weight of the control group, separate for sexes) were not included.
Cohort 1A: One male and one female/litter (20/sex/group)
Cohort 1B: One male and one female/litter (25/sex/group)
Selected F1 offspring received the test substance daily by gavage until one day before sacrifice.

Standardization of litters (F1 generation pups):
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 5 male and 5 female pups (always the first 5 pups/sex and litter were taken for further rearing). If individual litters did not have 5 pups/sex, the litters were processed in such a way that the most evenly distributed 10 pups per litter were present for further rearing (e.g., 6 male and 4 female pups). Surplus animals were sacrificed. Standardization of litters was not performed in litters with ≤ 10 pups.

Pups after standardization/weaning:
With the exception of those F1 generation pups, which were chosen as F1 rearing animals, and those F1 pups, which were chosen for blood sampling on PND 4 and 22, all pups were sacrificed under isoflurane anesthesia with CO2 after standardization or weaning. The pups chosen for blood sampling were sacrificed by decapitation under isoflurane anesthesia.
All culled pups, including stillborn pups and those that died during their rearing period, were subjected to a macroscopic (external and visceral) examination.
All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were further evaluated on a case-by-case basis (e.g., histopathological evaluation or special staining), depending on the findings noted.

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

test group 01 (parental animals), test group 11 (cohort 1A and 1B animals)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

test group 02 (parental animals), test group 12 (cohort 1A and 1B animals)

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

test group 03 (parental animals), test group 13 (cohort 1A and 1B animals)

No. of animals per sex per dose:

F0: 25 animals per sex per dose
F1A: 20 animals per sex per dose
F1B: 25 animals per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a previous OECD No. 414 study (BASF, project No. 30R0333/03R011, 2016), Citronellylnitril was administered as an oily solution to groups of 25 time-mated female rats by gavage at doses of 50, 150 and 450 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. Maternal toxicity was observed at the highest dose level of 450 mg/kg bw/d, namely increased absolute and relative liver weights (19 and 21% above control) and corresponding clinical-pathological changes (increased triglyceride and cholesterol values). Therefore, the no observed adverse effect level (NOAEL) for maternal toxicity was set to 150 mg/kg bw/d.
In a previous OECD No. 408 study (SPL, project No. 2153-0004, 2008), Citronellylnitril was administered by oral gavage at dose levels of 300,100, 30, 10 and 0 mg/kg bw/d for a period of ninety consecutive days. No adverse effects were observed in clinical examinations. In (histo)pathology, centrilobular, hepatocyte enlargement and increased absolute and relative liver weights at 300 (and partly at 100) mg/kg bw/d were observed. These changes were discussed as adaptive and the NOAEL was set to 300 mg/kg bw/d.
In an OECD No. 415 one-generation reproduction study in rats with an evaluation through sexual maturity in the F1 Generation (CLR, study No. TIF00044, 2011), Citronellylnitril was administered by oral gavage to 25 rats per sex and group at dose levels of 500, 200, 75 and 0 mg/kg bw/d. The purpose of this study was to provide information concerning the effects of Citronellylnitrile on gonadal function, estrous cycles, mating behavior, conception, parturition, lactation and the growth and development of offspring up to day 60 postpartum.
In this study, the no-observable-adverse-effect-level (NOAEL) for systemic toxicity was 200 mg/kg/day based on the below mentioned effects. At 500 mg/kg/day, reductions in body weight gains occurred in F0 male rats prior to cohabitation, followed by persistent reductions in weight gain through the end of the study (10% below control). In pathology, increased absolute and relative brain (males: 4 and 11% above control), spleen (males: 17 and 25% above control, respectively), liver (males: 35 and 44%; females: 15 and 9% above control) and kidney weights (males: 5 and 14% above control) occurred in F0 animals at 500 mg/kg/d. Microscopic findings that could be correlated with the changes in organ weights were not observed. The NOAELs for reproductive performance in the F0 and for viability and growth of the F1 generation offspring was greater than 500 mg/kg/d.
Based on the above-mentioned study results, the following dose levels were selected: 25 mg/kg bw/d as low dose, 100 mg/kg bw/d as intermediate dose, 400 mg/kg bw/d as high dose.
- Fasting period before blood sampling for clinical biochemistry:
yes, for about 16-20 hours

Positive control:

not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Mortality: twice daily on working days or once daily (Saturday, Sunday or on public holidays).
Cage side observations: at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
F0: animals once before the administration and subsequently once per week
F1 (Cohort 1A/1B): at weekly intervals during the administration period.

BODY WEIGHT: Yes
- Time schedule for examinations: once a week, except
• during the mating period of the F0 and F1 parental animals, the females were weighed on the day of positive evidence of sperm GD 0 and on GD 7, 14 and 20.
• females with litter were weighed on the day of parturition PND 0 and on PND 1, 4, 7, 10, 14, 18 and 21.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Generally, food consumption was determined once a week (over a period of 7 days) for male and female F0 parental animals and F1 rearing animals, with the following exceptions:
• not determined after the 10th premating week (male F0 animals) and during the mating period (male and female F0 animals).
• during pregnancy, food consumption of the F0 females with evidence of sperm was determined weekly for GD 0-7, 7-14 and 14-20.
• during lactation, food consumption of the F0 females, which gave birth to a litter was determined for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in the females after weaning.

WATER CONSUMPTION: No

CLINICAL PATHOLOGY
Samples were withdrawn from 10 F0 parental males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined.

Hematology
Parameters examined:
- Leukocyte count (WBC)
- Erythrocyte count (RBC)
- Hemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RETA)
- Prothrombin time (Hepato Quick’s test; HQT)

Clinical chemistry
Parameters examined:
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)
- Alkaline phosphatase (ALP)
- gamma-Glutamyltransferase (GGT)
- Sodium (NA)
- Potassium (K)
- Chloride (CL)
- Inorganic phosphate (INP)
- Calcium (CA)
- Urea (UREA)
- Creatinine (CREA)
- Glucose (GLUC)
- Total bilirubin (TBIL)
- Total protein (TPROT)
- Albumin (ALB)
- Globulins (GLOB)
- Triglycerides (TRIG)
-Cholesterol (CHOL)

Hormones:
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits. T4 was examined via ELISA.

Urinalysis
Parameters examined:
- pH
- Protein (PRO)
- Glucose (GLU)
- Ketones (KET)
- Urobilinogen (UBG)
- Bilirubin (BIL)
- Blood
- Specific gravity
- Sediment
- Color, turbidity (COL, TURB)
- Volume (VOL)

Oestrous cyclicity (parental animals):

Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental animals for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.

At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

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 sacrificed on schedule:
- Sperm motility
- Sperm morphology
- Sperm head count (cauda epididymis)
- Sperm head count (testis)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes

PARAMETERS EXAMINED
- Pup viability/mortality:
twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21.

- Sex ratio
determined on the day of birth (PND 0) by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.

- Pup clinical observations
examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams.

- Nipple/areola anlagen
examined and counted in all surviving male pups on PND 13 and on PND 20.

- Pup body weight data
on the day after birth (PND 1) and on PND 4 (before standardization), 7, 14 and 21.

- Anogenital distance
Anogenital distance (AGD) is defined as the distance from the center of the anal opening to the base of the genital tubercle. AGD was determined in all live male and female pups on PND 1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.

- Vaginal opening
All female F1 pups selected to become the F1 rearing animals (cohort 1A and 1B) were evaluated daily for vaginal patency beginning on PND 27. On the day of vaginal opening the body weights of the respective animals were determined.

- Preputial separation
All male F1 pups selected to become the F1 rearing animals (cohort 1A and 1B) were evaluated daily for preputial separation beginning on PND 38. On the day of preputial separation the body weights of the respective animals were determined.

CLINICAL PATHOLOGY (Cohort 1A):
Samples were withdrawn from 10 cohort 1A males and females per group at termination. Blood samples were taken from animals by puncturing the retrobulbar venous plexus following isoflurane anesthesia. In the afternoon preceding the day of urinalysis, the animals were individually transferred into metabolism cages (no food or drinking water provided); on the following morning, the individual urine specimens were examined.

Hematology
Parameters examined:
- Leukocyte count (WBC)
- Erythrocyte count (RBC)
- Hemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RETA)
- Prothrombin time (Hepato Quick’s test; HQT)

Clinical chemistry
Parameters examined:
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)
- Alkaline phosphatase (ALP)
- gamma-Glutamyltransferase (GGT)
- Sodium (NA)
- Potassium (K)
- Chloride (CL)
- Inorganic phosphate (INP)
- Calcium (CA)
- Urea (UREA)
- Creatinine (CREA)
- Glucose (GLUC)
- Total bilirubin (TBIL)
- Total protein (TPROT)
- Albumin (ALB)
- Globulins (GLOB)
- Triglycerides (TRIG)
- Cholesterol (CHOL)

Urinalysis
Parameters examined:
- pH
- Protein (PRO)
- Glucose (GLU)
- Ketones (KET)
- Urobilinogen (UBG)
- Bilirubin (BIL)
- Blood
- Specific gravity
- Sediment
- Color, turbidity (COL, TURB)
- Volume (VOL)

Hormone analysis (F1; PND 4 and PND 22)
Blood samples were withdrawn from 10 surplus (culled) PND 4 offspring (as far as possible of different litters) per sex and group. PND 4 samples were pooled per sex and litter if the available amount is not sufficient for a hormone analysis. Blood samples were withdrawn from 10 surplus PND 22 offspring (as far as possible of different litters) per sex and group. The blood samples were collected after decapitation (following isoflurane anesthesia) from the Vena cava cranialis.
Parameters examined:
- Total thyroxine (T4)
- Thyroid stimulating hormone (TSH)
The concentrations of TSH were determined by radioimmunoassay (RIA), using commercially available RIA test kits. T4 was examined via ELISA.

OESTROUS CYCLICITY (F1 ANIMALS)
In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle was also evaluated in cohort 1A and 1B females for 2 weeks around PND 75.
An internal peer review of the estrous cycle data prepared around PND 75 including all vaginal smears taken from cohorts 1A and 1B female animals of test group 0 (0 mg/kg bw/d) was performed because the estrous cycle data of the control group (e.g. cycle length) was outside the range of the historical control data of the test facility.
In general, the original and peer reviewed estrous cycle data was comparable.
At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each cohort 1A and 1B female with scheduled sacrifice.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

SACRIFICE
All F0 generation parental animals were sacrificed by decapitation under isoflurane anesthesia.

GROSS NECROPSY
The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.

ORGAN WEIGHTS
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Ovaries
10. Pituitary gland (fixed)
11. Prostate (ventral and dorsolateral part together, fixed)
12. Testes
13. Seminal vesicles including coagulating glands (fixed)
14. Spleen
15. Thymus (fixed)
16. Thyroid glands (with parathyroid glands) (fixed)
17. Uterus with cervix
All paired organs were weighed together (left and right).


HISTOPATHOLOGY
The organs or tissues were fixed in 4% neutral-buffered formaldehyde solution except for epididymis, testis, eyes and ovaries (fixed in modified Davidson’s solution):
Organs/ tissues examined:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left
11. Esophagus
12. Eyes with optic nerve
13. Heart
14. Ileum
15. Jejunum
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic, lumbar)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left
35. Thymus
36. Thyroid glands
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas deferens

Animals that died or were sacrificed in a moribund state were processed histotechnically and assessed like control animals.

The ovaries and eyes with optic nerve of animals that died or were sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution.

The left testis and left epididymis of all male F0 parental animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis.

For technical reasons, the left testis, left epididymis, and the eyes with optic nerves of all animals of the F0 parental control and high dose groups sacrificed at scheduled dates were embedded in paraplast after fixation. For the same reason, the ovaries of all F0 females in all test groups were embedded in paraplast.

In case of macroscopic findings in the right testis or right epididymis, this testis as well as the corresponding epididymis were fixed for histopathological examination and the left testis and epididymis were used for sperm analysis.

The uteri of all cohabited female F0 generation parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method, Salewski 1964). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl).


Subcontraction of histotechnical processing
With the exception of intermediate test groups, the histotechnical processing (cutting and HE staining) of all organs of all F0 parental animals and the histotechnical processing (paraplast embedding, cutting, HE staining) of all organs of all cohort 1A animals (according to study plan, all amendments and raw data) was accomplished by the test site TPL Path Labs GmbH (TPL) Sasbacher Straße 10, 79111 Freiburg; Germany. The ovaries of both the F0 generation animals, were processed at the test facility and were not sent to TPL.
The study phase histotechnical processing was performed under the test site code 1701/20.
Raw data of the study phase, as well as wet tissues, paraplast blocks and HE-stained slides were sent to the test facility for archiving for at least the period of time specified in the GLP principles.

Intermediate test groups:
The thyroid and liver of test group 01 and 02 male animals (F0) were processed histotechnically at BASF SE.

Histopathological evaluation of the HE-stained slides was performed by the study pathologist.

Peer Review
After completion of the histopathological assessment by the study pathologist an internal peer review including liver and thyroid glands of all examined male animals of the F0 parental generation and the spleen of all male animals of the F1, rearing animals, cohort 1A was performed (BASF SE, Ludwigshafen). Results presented in the pathology report reflect the consensus opinion of the study pathologist and the peer review pathologist.

Postmortem examinations (offspring):

SPERM PARAMETERS (Cohort 1A)
After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male cohort 1A animals sacrificed on schedule:
- Sperm motility
- Sperm morphology
- Sperm head count (cauda epididymis)
- Sperm head count (testis)

DOFC (Cohort 1A)
A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (cohort 1A females) according to Plowchalk et.al. (1993). In general, sections were prepared with 2 - 3 µm thickness and serial sections were taken every 100 µm to complete up to 15 – 20 cut levels across the whole ovarian tissue. For the counting of primordial and growing follicles, H&E stained slides were prepared from all cut levels.


PATHOLOGY (F1 pups)
All surplus F1 generation pups that were not used for the following organ weight determinations were sacrificed under isoflurane anesthesia with CO2. The selected pups for organ weight determination were sacrificed by decapitation under isoflurane anesthesia. All animals were necropsied and assessed by gross pathology with special emphasis on the reproductive organs.

- Organ weights
The following weights were determined in up to 10 animals per sex per group sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Brain
3. Spleen
4. Thymus (fixed)

- Organ/ tissue fixation
The following organs or tissues of up to 10 animals per sex per group were fixed in 4% neutral-buffered formaldehyde solution:
1. All gross lesions
2. Brain
3. Mammary gland (male and female)
4. Spleen
5. Thymus
6. Thyroid glands


PATHOLOGY (Cohort 1A)
All F1 generation rearing animals, cohort 1A were sacrificed by decapitation under isoflurane anesthesia.

- Gross neropsy (Cohort 1A)
The exsanguinated animals were necropsied and assessed by gross pathology.

- Organ weights (Cohort 1A)
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only)
11. Ovaries
12. Pituitary gland (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes
15. Seminal vesicles including coagulating glands (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus with cervix
All paired organs were weighed together (left and right).

- Histopathology (Cohort 1A)
The organs or tissues were fixed in 4% neutral-buffered formaldehyde solution except for epididymis, testis, eyes and ovaries (fixed in modified Davidson’s solution):
Organs/ tissues examined:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left
11. Esophagus
12. Eyes with optic nerve
13. Heart
14. Ileum
15. Jejunum
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left (fixed in modified Davidson´s solution)
35. Thymus
36. Thyroid glands (with parathyroid glands)
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas (ductus) deferens

Animals that died or were sacrificed in a moribund state were processed histotechnically and assessed like control animals.

The ovaries and eyes with optic nerve of animals that died or were sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution.

For technical reasons, the ovaries of all cohort 1A females in all test groups were embedded in paraplast.

The left testis and left epididymis of all Cohort 1A animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis.

In case of macroscopic findings in the right testis or right epididymis, this testis as well as the corresponding epididymis were fixed for histopathological examination and the left testis and epididymis were used for sperm analysis.


Subcontraction of histotechnical processing
With the exception of intermediate test groups as listed below, the histotechnical processing (cutting and HE staining) of all organs of all F0 parental animals and the histotechnical processing (paraplast embedding, cutting, HE staining) of all organs of all cohort 1A animals (according to study plan, all amendments and BASF raw data) was accomplished by the test site TPL Path Labs GmbH (TPL) Sasbacher Straße 10, 79111 Freiburg; Germany. The ovaries of both the F0 generation animals and the Cohort 1A animals, were processed at the test facility and were not sent to TPL.
The study phase histotechnical processing was performed under the test site code 1701/20.
Raw data of the study phase, as well as wet tissues, paraplast blocks and HE-stained slides were sent to the test facility for archiving for at least the period of time specified in the GLP principles.

Intermediate test groups:
The spleen of test group 11 and 12 male animals (F1, cohort 1A) were processed histotechnically at BASF SE.

Histopathological evaluation of the HE-stained slides was performed by the study pathologist.


PATHOLOGY (Cohort 1B)
All cohort 1B were sacrificed by decapitation under isoflurane anesthesia.

- Gross neropsy (Cohort 1B)
The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.

- Organ weights (Cohort 1B)
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Cauda epididymis
4. Epididymides
5. Liver
6. Ovaries
7. Pituitary gland (fixed)
8. Prostate (ventral and dorsolateral part together, fixed)
9. Testes
10. Seminal vesicles including coagulating gland (fixed)
11. Uterus (with cervix)

- Histopathology (Cohort 1B)
The organs or tissues were fixed in 4% neutral-buffered formaldehyde solution except for epididymis, testis and ovaries (fixed in modified Davidson’s solution):
Organs/ tissues examined:
1. All gross lesions
2. Adrenal glands
3. Cervix uteri
4. Coagulating glands
5. Epididymis, left
6. Liver
7. Ovaries
8. Pituitary gland
9. Prostate
10. Seminal vesicles including coagulating glands
11. Testis
12. Uterus
13. Vagina

Histotechnical processing and examination by light microscopy was not performed

For technical reasons, the ovaries of all cohort 1B females of all test groups were embedded in paraplast.


Peer Review
After completion of the histopathological assessment by the study pathologist an internal peer review including liver and thyroid glands of all examined male animals of the F0 parental generation and the spleen of all male animals of the F1, rearing animals, cohort 1A was performed (BASF SE, Ludwigshafen). Results presented in the pathology report reflect the consensus opinion of the study pathologist and the peer review pathologist.

Statistics:

- DUNNETT test (two-sided): Food consumption (parental and rearing animals), body weight and body weight change (parental animals, pups and rearing animals; for the pup weights, the litter means were used), estrous cycle duration, number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter, duration of sexual maturation (days to vaginal opening, days to preputial separation), anogenital distance, anogenital index
- FISHER'S EXACT test (one-sided): Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy, sexual maturation data (vaginal opening, preputial separation)
- WILCOXON-test (one-sided): Presence of areolae/nipples, proportions of affected pups per litter with necropsy observations, urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity)
- WILCOXON-test (one-sided) with BONFERRONI-HOLM adjustment: Spermanalysis parameters
- WILCOXON-test (one-sided-): DOFC
- KRUSKAL-WALLIS test (two-sided) + WILCOXON-test (two-sided): Organ weight parameters
- KRUSKAL-WALLIS test + WILCOXON-test (one or two-sided): Blood parameters, Urine pH, volume and specific gravity

Reproductive indices:

Male mating index (%) = number of males with confirmed mating / number of males placed with females x 100

Male fertility index (%) = number of males proving their fertility / number of males placed with females x 100

Female mating index (%) = number of females mated / number of females placed with males x 100

Female fertility index (%) = number of females pregnant / number of females mated x 100

Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x 100

Offspring viability indices:

Live birth index (%) = number of liveborn pups at birth / total number of pups born x 100

Postimplantation loss (%) = number of implantations – number of pups delivered / number of implantations x 100

Viability index (%) = number of live pups on day 4* after birth / number of live pups on the day of birth x 100
* before standardization of litters (i.e. before culling)

Lactation index (%) = number of live pups on day 21 after birth / number of live pups on day 4** after birth x 100
**after standardization of litters (i.e. after culling)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse findings were observed.

Clinical observations for males and females (except gestation and lactation period)
Transient salivation during a short time period after gavage dosing (up to 2 hours) was noted for all male and female animals of test group 03 (400 mg/kg bw/d) and six males of test group 02 (100 mg/kg bw/d) during the entire study period. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.
Male animal No. 33 of test group 01 (25 mg/kg bw/d) showed an unsteady gait from study day 35 onwards, starting between 2 -5 h after application on study day 35. One female animal No. 175 of test group 02 (100 mg/kg bw/d) showed respiration sounds from study day 74 onwards as well as during the mating period. These individual observations were not related to dose
and, therefore, not assessed as treatment-related.
Note: On pre-mating day 38, the clinical examinations between 2-5 hours after treatment were not performed for all animals of test groups 00-03. Thus, no documentation exists in the electronic system and are not shown in tables. These missing records do not have an influence on the validity of the present study.

Clinical observations for females during gestation of F1 litters
Transient salivation during a short time period after gavage dosing (up to 2 hours) was noted in female animals of test group 03 (400 mg/kg bw/d) during the entire gestation period. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.

Clinical observations for females and offspring during lactation of F1 litters
Transient salivation (please see above) in female animals of test group 03 (400 mg/kg bw/d) was still present during lactation.
Female animal No. 141 of test group 01 (25 mg/kg bw/d) showed a swelling on the right side at the mammary line during PND 14 and 24. Female animal No. 125 of test group 00 (control group, 0 mg/kg bw/d) as well as female animal No. 158 of test group 02 (100 mg/kg bw/d) had no more pups alive on PND 0 (complete litter loss). These observations were not considered to be associated with the test compound since there were not related to dose.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test substance-related mortalities in any of the groups.
One female animal (No. 176) of test group 03 (400 mg/kg bw/d) was found dead on PND 3. The death of this animal was not preceded by any specific preterminal clinical signs. This isolated death of one single animal was assessed as spontaneous and not related to treatment.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights as well as mean body weight change values of all test substance-treated male and female F0 animals were comparable to the concurrent control values throughout the entire study.
In male animals of test group 03 (400 mg/kg bw/d) the mean body weight change value was significantly decreased during premating days 56 – 63. The mean body weight change values in female animals of test group 03 (400 mg/kg bw) were also significantly decreased during premating days 49 – 56 and 63 - 70. However, during premating days 56 – 63 the mean body weight change value in female animals of test group 03 (400 mg/kg bw/d) was significantly increased. The body weight change values were decreased/increased during a short time period and are within the normal range of biological variation. Therefore, these findings were considered to be spontaneous in nature and not treatment related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In general, mean food consumption of all male and female animals of all test groups was comparable to the concurrent control values throughout the entire study. In test group 03 (400 mg/kg bw/d), food consumption was significantly increased in male animals on study day 35 (20% above control) and in female animals on PND 21 (14 % above control). Since all other intervals were comparable to control, these slight temporary increases were assessed as spontaneous and not related to treatment.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No treatment-related, adverse changes among hematological parameters were observed.
At the end of the administration period, in parental females of test groups 02 and 03 (100 and 400 mg/kg bw/d) absolute monocyte counts were significantly increased. However, this was the only altered hematology parameter among these individuals. Therefore, it was regarded as treatment related but non-adverse (ECETOC Technical Report No. 85, 2002).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of the administration period, in parental females of test group 03 (400 mg/kg bw/d) triglyceride levels were significantly increased. This alteration in combination with increased liver weights was regarded as a treatment-related and adverse effect (ECETOC Technical Report No. 85, 2002).

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

In parental males (test groups 01, 02 and 03; 25, 100, 400 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.
In F0 females of test group 03 (400 mg/kg bw/d), T4 values were significantly decreased. However, the T4 mean was within the historical control range, whereas that one of the study controls was above this range (F0 females, T4 24.28-43.26 nmol/L). Therefore, this alteration was 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.
At the end of the administration period, the urine pH value in males and females of test group 03 (400 mg/kg bw/d) as well as in females of test group 02 (100 mg/kg bw/d) was significantly decreased. Additionally, in males of test group 03, urine volume was significantly decreased, and urine specific gravity was significantly increased. Among these individuals the levels of ketone bodies in the urine und the numbers of granular and epithelial cell casts in the urine sediment were significantly increased. However, all mentioned changes were observed without relevant changes in other clinical pathology parameters and without any histopathological change in the kidneys. Therefore, these changes were regarded as maybe treatment related but non-adverse.
In parental females of test groups 02 and 03 (100 and 400 mg/kg bw/d), urine specific gravity was significantly increased and in females of test group 02 urine volume was significantly decreased. However, these changes were not dose dependent. Therefore, they were regarded as incidental and not treatment related.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were noted in liver and thyroid in male animals of test group 03.

Liver
In the liver, an increased incidence of minimal centrilobular hepatocellular hypertrophy was observed, characterized by minimally increased size of the cytoplasm.
- Hypertrophy, centrilobular, grade 1 (males): 1 / 1 / 0 / 11 in control, low, mid and high dose groups, respectively

Thyroid glands
In the thyroid glands, there was an increased incidence of minimal follicular
hypertrophy/hyperplasia characterized by a high prismatic shape of cells and smaller size of follicular lumina.
- Hypertrophy/hyperplasia, follicular cell, grade 1 (males): 2 / 2 / 2 / 10 in control, low, mid and high dose groups, respectively

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.

Fertility
The female animals (No. 108, 144), which were not pregnant as well as one of the male mating
partners (No. 44) did not show relevant histopathological findings consistent with impaired
fertility.
Male animal No. 8 showed marked tubular degeneration in the left testis and severe
oligospermia bilateral in the epididymides, which was considered the cause for the lack of
offspring of the mating pair of animals 8/108. This was considered a spontaneous occurrence
in the control group.
Decedents
The female animal No. 176 was found dead 104 days after start of exposure. No macroscopic
or microscopic findings were observed that could explain the death of this animal.

Histopathological findings: neoplastic:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration was comparable between the groups: 4.0 / 4.1 / 4.1 and 4.0 days in test groups 00 - 03, respectively.

Reproductive function: sperm measures:

no effects observed

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 of parental males, no treatment-related effects were observed.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups (00 - 03).
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
Male animal No. 8 of test group 00 (control; 0 mg/kg bw/d) as well as male animal No. 44 of test group 01 (25 mg/kg bw/d) did not generate F1 pups.
Thus, the male fertility index ranged between 96% and 100%, reflecting the normal range of biological variation inherent in the strain of rats used for this study.

The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until copulation was detected (GD 0) varied between 2 and 3 days without any relation to dosing.
All female rats delivered pups or had implants in utero with the following exception:
• Test group 00 (control, 0 mg/kg bw/d): Female No. 108 (mated with male No. 8) did not become pregnant
• Test group 01 (25 mg/kg bw/d): Female No. 144 (mated with male No. 44) did not become pregnant
These animals did not show relevant gross or histopathological findings consistent with impaired fertility, except of control animal No. 8.
The fertility index ranged between 96% and 100% without showing any relation to dosing.
The mean duration of gestation was comparable in all test groups (i.e. between 22.3 and 22.6 days).
The gestation index ranged between 95.8% and 100% without showing any relation to dosing.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.7 / 12.7 / 13.7 and 12.3 implants/dam in test groups 00 - 03, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (11.6 / 10.2 / 10.5 and 4.1 mean% in test groups 00 - 03, respectively), and the mean number of F1 pups delivered per dam remained unaffected (12.2 / 11.4 / 12.2 and 12.1 pups/dam, respectively in test groups 00 - 03).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 97% / 97% / 97% and 99% in test groups 00 - 03. Moreover, the number of stillborn pups was not significantly different between the test groups.

Key result

Dose descriptor:

NOAEL

Remarks:

fertility and reproductive performance

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Increased triglyceride values in combination with increased liver weights

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F1 generation pups/litters
There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
Male animal No. 134-02 of test group 01 (25 mg/kg bw/d) showed a poor general condition as well as a reduced nutritional condition on PND 8. Therefore, this animal was sacrificed moribund on PND 8. Further, reduced nutritional condition was observed in male animal No. 135-09 of test group 01 (25 mg/kg bw/d) on PND 1. Closed eyelids was detected in male animal 183-03 of test group 03 (400 mg/kg bw/d) from PND 19 onwards. Since all these findings occurred in individual pups mostly without relation to dose, they were not considered to be treatment-related.


F1 rearing animals, Cohort 1A
Transient salivation during a short time period after gavage dosing (up to two hours) was noted for all male and female animals of test group 13 (400 mg/kg bw/d) during the entire study. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any treated male and female animals of all test groups 11, 12 and 13 (25, 100 and 400 mg/kg bw/d).
The findings in control group were assessed as spontaneous in nature, i.e. male animal. No. 216 showed a small testis on the right side from study day 14 to 34 and female animal No. 303 showed a kinked tail from study day 14 onwards.

No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
Male animal. No. 216 of the control group (Test group 10, 0 mg/kg bw/d) showed a small testis on the right side from study day 14 to 34. This finding was also seen on the respective days of detailed clinical observation, i.e. study days 14, 21 and 28. Further, female animal No. 303 of the control group (Test group 10, 0 mg/kg bw/d) showed a kinked tail from study day 14 onwards, which was also seen on the respective days of detailed clinical observation from study day 14 onwards. These findings in the control group were assessed as spontaneous in
nature.


F1 rearing animals, Cohort 1B
No treatment-related, adverse clinical signs were observed.
Transient salivation during a short time period after gavage dosing (up to 2 hours) was noted for all male and female animals of test group 13 (400 mg/kg bw/d) during the entire study period starting on study day 2. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.
An anomaly of teeth was observed in male animal No. 447 of test group 11 (25 mg/kg bw/d) from study day 35 to 57. This finding was considered to be spontaneous in nature and not related to test substance treatment.
Note: On study day 7, the clinical examinations between 2-5 hours after treatment were not performed for all animals of test groups 10 – 13 in cohort 1B. Thus, no documentation exists in the electronic system and are not shown in tables. These missing records do not have an influence on the validity of the present study.

No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
An anomaly of teeth was observed in male animal No. 447 of test group 11 from study day 35 to 57 as well as on the respective days of detailed clinical observation, i.e. study days 35, 42, 49 and 56. This finding was considered to be spontaneous in nature.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1 generation pups/litters
The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99% / 98% / 98% and 93% in test groups 00 – 03 (0, 25, 100 and 400 mg/kg bw/d).
Female animal No. 176 of test group 03 was found dead on PND 3. Therefore, all remaining pups of that animal were sacrificed on PND 3. This led to a viability index of 93% in test group 03. However, it was not assessed as related to treatment.
The lactation index indicating pup survival on PND 4 - 21 was 100% / 99% / 100% and 100% in test groups 00 - 03.


F1 rearing animals, Cohort 1A
There were no test substance-related mortalities in any of the groups.
Female animal No. 380 of test group 13 (400 mg/kg bw/d) was found dead on study day 26. The death of this animal was not preceded by any specific preterminal clinical signs. Therefore, the death was assessed as spontaneous and not related to treatment.


F1 rearing animals, Cohort 1B (03S032_1B)
There were no test substance-related mortalities in any of the groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

F1 generation pups/litters
In general, the mean body weights and body weight change values of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study.
On PND 1, mean body weights of female animals as well as female and male animals combined of test group 01 (25 mg/kg bw/d) were significantly increased. In addition, the mean body weight of female animals of test group 01 (25 mg/kg bw/d) was significantly increased on PND 4. Since no dose-response relationship occurred, these isolated findings were considered to be spontaneous in nature and not related to treatment.


F1 rearing animals, Cohort 1A
In general, no treatment-related, adverse findings in body weight (change) were observed. In males of test group 13, mean body weight was slightly decreased towards the end of the treatment period (6% below control, on study day 63). Consistently, body weight change was significantly decreased during study days 0 – 63. This was assessed as treatment-related but not adverse since the level of decrease was slight. Females of test group 13 showed a mean body weight comparable to control values.
The following findings were not related to dose and, therefore, not assessed as treatment related.
Female animals of test group 12 showed a significant increase in body weight on study days 0 and 7.
In males of test group 13 and 12, mean body weight change values were significantly decreased during study days 42 – 49, without a dose-response relationship.


F1 rearing animals, Cohort 1B
In general, mean body weight and mean body weight change values of all male and female animals of test groups 11, 12 and 13 (25, 100, 400 mg/kg bw/d) were comparable to the concurrent test group 10 (control group; 0 mg/kg bw/d).
In male animals of test group 13, body weight change was significantly decreased during study days 42 – 49. Since test group 13 males gained weight comparable to control during the entire study phase, this was assessed as not related to treatment.
All other findings observed in the test groups, e.g. increased body weight of test group 12 females on study day 21, increased body weight change of test groups 12 and 13 females during study days 14 – 21 and decreased body weight change of test group 11 males during 14 – 21, showed no relation to dose and were assessed to be spontaneous.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1A
In general, mean food consumption was comparable to the concurrent control values in all test groups over the entire study period.
In test group 11, males and females showed a significant decrease in mean food consumption during study days 49 - 56 and 42 - 56, respectively. Since this was not related to dose, it was not assessed as treatment-related.


F1 rearing animals, Cohort 1B
In general, food consumption of all male and female animals of all test groups was comparable to the concurrent control values throughout the entire study.
In test group 13, food consumption was significantly increased in males during study days 21 - 28 and 35 – 42 (each 8% above control) and in females during study days 21 - 28 (8% above control). However, this minor increase was only temporary during one or two intervals. Therefore, the findings were not assessed as treatment-related.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At PND90 in F1 males of test group 13 (400 mg/kg bw/d) absolute reticulocyte counts were significantly increased. This change in combination with increased spleen weights and increased gradings of extramedullary hematopoiesis was regarded as treatment related and adverse (ECETOC Technical Report No. 85, 2002).
Additionally, in males of this test group absolute and relative eosinophil counts were significantly decreased. Eosinophil count decreases were isolated, without any change of other differential blood cell parameters. Therefore, also this change was regarded as treatment related but non-adverse (ECETOC Technical Report No. 85, 2002).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among clinical chemistry parameters were observed.
At PND90 in males of test group 13 (400 mg/kg bw/d), cholesterol values were significantly decreased. However, this was an isolated change without any other clinical chemistry alterations. Therefore, this change was regarded as treatment related but non-adverse.
In F1A females of test group 11 (25 mg/kg bw/d), alkaline phosphatase (ALP) activities were significantly increased but this change was not dose dependent. Therefore, it was 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.
Urine pH values in males and females of test group 13 (400 mg/kg bw/d) were significantly decreased. Additionally, in females of this test group urine volume was decreased (not statistically significantly). These changes reflect the adaptation of the kidneys towards less fluid income as well as the physiological regulation of urine pH value keeping the homeostasis of the blood pH value. Therefore, these changes were regarded as treatment related but adaptive rather than adverse.
In F1A males of test group 11 (25 mg/kg bw/d) urine pH values were also decreased, but the change was not dose dependent and therefore, it was regarded as incidental and not treatment related.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

Vaginal opening
Each female F1 pup, which was selected to become a rearing female, was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 27, the last was PND 36. The mean number of days to reach the criterion in the control and 25, 100 and 400 mg/kg bw/d test groups was 31.9; 31.6; 31.6 and 32.6 days, respectively. The mean body weight on the day, when vaginal opening was recorded, was 97.4, 96.8, 98.4 and
101.8 g in test groups 00 – 03, respectively. Neither a statistically significant nor a toxicologically relevant effect was noted in any of the treatment groups.

Preputial separation
Each male F1 pup, which was selected to become a rearing male, was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 38, the last was PND 44. The mean number of days to reach the criterion in the control and 25, 100 and 400 mg/kg bw/d test groups was 39.6, 40.0, 40.3 and 40.4 days, respectively. The mean body weight on the day, when preputial separation was recorded, was 162.5, 160.6, 163.5 and 162.5 g in test groups 00 – 03, respectively. Statistical significance was observed in
test groups 03 and 02. However, their mean values are close to the mean value of the historical control data (HCD, preputial separation: mean of means: 41.9 days). Therefore, this was not assessed as treatment-related, adverse finding.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The apparent number and percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13 as well as on PND 20.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Rearing animals, cohort 1A
Absolute organ weights
When compared with the control group 10 (=100%), the following mean absolute weights were significantly changed:
- Kidneys (females): 101% / 104% / 110%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 97% / 107% / 1113%* versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 10.

Relative organ weights
When compared with control group 10 (=100%), the following mean relative weights were significantly changed:
- Kidneys (males): 101% / 101% / 110%** versus ctrl in low, mid and high dose groups, respectively
- Liver (males): 98% / 99% / 111%* versus ctrl in low, mid and high dose groups, respectively
- Spleen (males): 99% / 100% / 107%* versus ctrl in low, mid and high dose groups, respectively
- Kidneys (females): 104% / 103% / 112%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 100% / 105% / 114%** versus ctrl in low, mid and high dose groups, respectively
- Spleen (females): 98% / 100% / 107%* versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 10.

The increased mean absolute and relative weights of the kidneys and liver of test group 13 male and female animals and the increased mean relative weights of the spleen of male test group 13 animals were regarded to be treatment-related.
The increased mean relative weight of the spleen of female test group 13 animals was regarded as incidental as there were no changes in histopathology or hematology parameters.
As described in amendment No. 3 to the study plan, five animals of each test group were sacrificed without prior fasting period. In males of test groups 10 and 13, vacuolation consistent with glycogen storage was noted in these animals. The weights of the livers of these five animals per group were approximately 1-5g higher than other animals of the respective groups which were fasted. In females, livers of the non-fasted animals were also heavier than other animals of the respective groups, albeit to a smaller degree than in males. Histopathologically,
these animals also showed vacuolation consistent with glycogen storage. As the magnitude of change was comparable in each group, this did not interfere with the interpretation of this study.


Rearing animals, cohort 1B
Absolute organ weights
When compared with the control group 10 (=100%), the following mean absolute weights were significantly changed:
- Liver (males): 96% / 105% / 108%* versus ctrl in low, mid and high dose groups, respectively
- Prostate (males): 91%* / 96% / 84%** versus ctrl in low, mid and high dose groups, respectively
- Testes (males): 98% / 99% / 92%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 99% / 103% / 109%** versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 10.

Relative organ weights
When compared with control group 10 (=100%), the following mean relative weights were significantly changed:
- Liver (males): 98% / 103% / 113%** versus ctrl in low, mid and high dose groups, respectively
- Prostate (males): 93% / 94% / 88%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 98% / 102% / 108%** versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 10.

The statistically significantly increased mean absolute and relative weights of the liver in test group 13 male and female animals were regarded to be treatment – related.
The mean absolute weights of the prostate were statistically significantly decreased in test groups 11 and 13. The weights of control (0.847g), test groups 01 (0.774g) and 12 (0.815g) were above the historical control range of 0.686 – 0.748g. The mean absolute weight of test group 13 (0.708g) was within the historical control range. The statistically significantly decreased mean relative weight of the prostate of test group 13 (0.228%) was within the historical control range of 0.213 – 0.231%, while all other test groups including controls lay above this range (test group 10: 0.26%, test group 11: 0.241%, test group 12: 0.246%). As there was no clear dose-response, and the weights of the prostate in cohort 1A were not significantly changed, these weight changes were regarded to be incidental.
The statistically significantly decreased mean absolute weight of the testes in test group 13 males (3.259g) was below the historical control range of 3.338 – 3.51g, while the control test group 10 (3.532g) was above this range. As there were no statistically significant changes in cohort 1A or the parental generation nor any treatment-related findings in spermanalysis or histopathology in cohort 1A and the parental generation, these changes were regarded to be
incidental.


Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts)
Absolute and relative organ weights
None of the mean absolute and relative weight parameters showed significant differences when compared to the control group 00.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Rearing animals, cohort 1A
All findings occurred either individually or were well-described spontaneous lesions (focal constriction/herniation of the liver in two test group 11 females (McInnes 2012). They were considered to be incidental or spontaneous in origin and without any relation to treatment.


Rearing animals, cohort 1B
All findings occurred either individually or were well-described spontaneous lesions (focal constriction/herniation of the liver in two test group 11 females (McInnes 2012). They were considered to be incidental or spontaneous in origin and without any relation to treatment.


Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts)
No gross lesions were observed.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

Rearing animals, cohort 1A
Increased hematopoiesis (reticulocytes) was noted in the spleen of male animals of test group 13.
- Hematopoiesis, extramedullary, grade 1 (males): 8 / 8 / 10 / 6 in control, low, mid and high dose groups, respectively
- Hematopoiesis, extramedullary, grade 2 (males): 6 / 3 / 3 / 6 in control, low, mid and high dose groups, respectively
- Hematopoiesis, extramedullary, grade 3 (males): 0 / 0 / 0 / 8 in control, low, mid and high dose groups, respectively

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.


Decedents
The female animal No. 380 was found dead 26 days after start of exposure. No macroscopic or microscopic findings were observed that could explain the death of this animal.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormones
In F1 PND4 and PND22 pups (test groups 01, 02 and 03; 25, 100, 400 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.
At PND90 in F1A males of test group 13 (400 mg/kg bw/d), T4 values were significantly decreased. However, in this test group T4 as well as TSH mean values were within historical control ranges (F1A males, T4 49.46-88.73 nmol/L, TSH 2.61-9.90 μg/L). Therefore, T4 change in males of test group 13 was regarded as incidental and not treatment related.
AT PND90 in F1A females of test groups 11, 12, and 13 (25, 100 and 400 mg/kg bw/d) no changes of T4 and TSH values were observed.

Spermanalysis:
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 of F1A males, no treatment-related effects were observed.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Differential ovarian follicle count
The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control group 10 and animals of test group 13

F1 rearing animals, Cohort 1A
Estrous cycle data generated for 2 weeks revealed regular cycles in the females of all test groups 10, 11, 12 and 13 (0, 25, 100 and 400 mg/kg bw/d). The mean estrous cycle duration was comparable between the groups: 5.1 / 4.4 / 5.3 and 4.3 in test groups 10 - 13, respectively.
The slightly higher mean value in the control and mid-dose group was assessed to be spontaneous and without relation to dose.

F1 rearing animals, Cohort 1B
Estrous cycle data generated for 2 weeks revealed regular cycles in females of all test groups 10 - 13. The mean estrous cycle duration in the different test groups was comparable: 5.4 / 4.5 / 4.4 and 4.8 days in test groups 10, 11, 12 and 13 (0, 25, 100 and 400 mg/kg bw/d), respectively.

Spermanalysis of F1 generation
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 of F1A males, no treatment-related effects were observed.

Thyroid hormones of F1 generation
In F1 PND4 and PND22 pups (test groups 01, 02 and 03; 25, 100, 400 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.
At PND90 in F1A males of test group 13 (400 mg/kg bw/d), T4 values were significantly decreased. However, in this test group T4 as well as TSH mean values were within historical control ranges (F1A males, T4 49.46-88.73 nmol/L, TSH 2.61-9.90 μg/L). Therefore, T4 change in males of test group 13 was regarded as incidental and not treatment related.
AT PND90 in F1A females of test groups 11, 12, and 13 (25, 100 and 400 mg/kg bw/d) no changes of T4 and TSH values were observed.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: increased absolute reticulocyte counts in combination with increased spleen weights and higher grades of extramedullary hematopoiesis (ncreased red blood cell metabolism)

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

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

In the chosen key study, a GLP compliant Extended One-Generation Reproduction Toxicity Study according to OECD guideline 443, Citronellylnitril was administered by gavage to groups of 25 male and 25 female Wistar rats at dose levels of 0 (test group 00), 25 (test group 01), 100 (test group 02) and 400 mg/kg body weight/day (mg/kg bw/d; test group 03). Corn oil served as vehicle. F0 animals were treated at least for 10 weeks prior to mating to produce litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts (1A and 1B) which were subjected to specific postweaning examinations. The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1A. Control animals were dosed daily with the vehicle only (corn oil).

In this extended one-generation reproduction toxicity study, adverse signs of systemic toxicity were observed at the high-dose level of 400 mg/kg bw/d. In the F0 parental female animals, increased triglyceride values in combination with increased liver weights indicated some slight alteration of the liver cell metabolism. F0 parental males of the high-dose group showed also liver effects, such as liver weight increases and minimal centrilobular hepatocellular hypertrophy, which were discussed as treatment-related but not adverse. Thus, under the conditions of this study, the NOAEL (no observed adverse effect level) for general, systemic toxicity in the F0 animals is the mid-dose level of 100 mg/kg bw/d) in females and the high-dose level of 400 mg/kg bw/d in males. In the F1A male offspring of high-dose group 13 (400 mg/kg bw/d), increased absolute reticulocyte counts in combination with increased spleen weights and higher grades of extramedullary hematopoiesis were signs of an increased red blood cell metabolism. F1A females showed no adverse signs of toxicity.

Thus, under the conditions of this study, the NOAEL (no observed adverse effect level) for general, systemic toxicity in the F1 animals is the mid-dose level of 100 mg/kg bw/d) in males and the high-dose level of 400 mg/kg bw/d in females.

The NOAEL for fertility and reproductive performance for the parental rats is 400 mg/kg bw/d), the highest tested dose.

The NOAEL for developmental toxicity in the F1 progeny is 400 mg/kg bw/d, the highest tested dose.

 

In addition. data from a GLP compliant one-generation reproduction study according to OECD guideline 415 is available. 100 rats per sex were assigned to four dosage groups, 25 rats per sex per group, for treatment with 3,7-dimethyloct-6-enenitrile (Charles River, 2011). Suspensions of 3,7-dimethyloct-6-enenitrile, or the vehicle, corn oil, were administered via gavage to the male rats once daily beginning 83 days before the cohabitation period, through the cohabitation period (maximum of 13 days) and continuing until the day before euthanasia; and to the female rats once daily beginning 14 days before the cohabitation period, through the cohabitation period (maximum of 13 days) and continuing through the day of euthanasia. Dosage levels were 0 (Vehicle), 75, 200 and 500 mg/kg bw/day. Surviving male rats were euthanized after completion of the cohabitation period. Female rats that delivered a litter and pups not selected for continued evaluation were euthanized on Day 22 of lactation (DL 22). Female rats that did not deliver a litter were euthanized on Day 25 of presumed gestation (DG 25). F1 generation rats selected for continued evaluation were euthanized on day 60 (± 3) postpartum.

The following effects were observed for P generation male rats: There were no treatment-related deaths at any dosage level tested. Clinical signs that were attributed to treatment with 3,7-dimethyloct-6-enenitrile included statistically significant increases in slight, moderate and extreme excess salivation at 500 mg/kg/day, and a low incidence of ungroomed coat in this group. At 500 mg/ kg bw /day, body weight gains for male rats were transiently, but significantly reduced during the first week of the dosage period (93% of controls), followed by non-significant reductions in body weight gains on DSs 15 to 22 (85% of controls), DSs 36 to 43 (86% of controls), DSs 78 to 84 (77% of controls), DSs 99 to 106 (68% of controls), DSs 106 to 113 (81% of controls) and DSs 113 to 120 (84% of controls). Body weight gains were again significantly reduced (65% of controls) on DSs 120 to 127 in this same dosage group, in comparison to the vehicle control group value. The sporadic reductions prior to cohabitation, and persistent reductions after cohabitation likely contributed to the overall significant reduction (90% of controls) in body weight gains that occurred at 500 mg/kg bw/day for the cumulative dosage period. Absolute and relative feed consumption values for P generation male rats were unaffected throughout the dosage period. There were no apparent effects of 3,7-dimethyloct-6-enenitrile on mating and fertility, reproductive organ weights or sperm motility and concentration at any dosage level tested. In addition, there were no treatment-related gross lesions or microscopic changes following 18 weeks of treatment at dosages up to and including 500 mg/ kg bw /day. At 500 mg/ kg bw /day, terminal body weights were reduced by 6%, in comparison to the vehicle control group value. In addition, statistically significant increases in the absolute and relative weights of the brain, liver, kidneys and spleen occurred in rats treated with 500 mg/ kg bw /day. However, there were no microscopic changes that could be correlated with the differences in brain, liver, kidney or splenic weights.

All P generation female rats survived until scheduled euthanasia. Similar to male rats, the number of female rats with excess salivation (slight and/or moderate) was significantly increased at 500 mg/kg bw/day, in comparison to the vehicle control group value. Body weights, body weight gains and absolute and relative feed consumption were unaffected by dosages of 3,7-dimethyloct-6-enenitrile as high as 500 mg/ kg bw /day prior to mating and during the gestation and lactation periods. In addition, there were no apparent effects on the estrous cycle, mating and fertility parameters or natural delivery and there were no treatment-related gross lesions or microscopic changes at any dosage level tested. Terminal body weights were significantly increased by 5% at 500 mg/ kg bw /day, as compared to the vehicle control group. Similar to P generation male rats, significant increases in the absolute and relative weights of the liver and kidneys occurred at 500 mg/ kg bw /day. There were no microscopic changes that could be correlated with the differences in liver or kidney weights. Microscopic examination of the ovaries in the 500 mg/ kg bw /day group, as compared to the vehicle control group, revealed that the ovaries of all female rats evaluated appeared to be functional with the presence of corpora lutea and growing follicles. A slight increase in the mean number of follicles per animal, along with higher variability, occurred at 500 mg/ kg bw /day compared to the female rats given the vehicle. Two rats in the 500 mg/kg/day dosage group showed comparatively high numbers of primordial follicles, most likely influencing both the noted increase from the vehicle control group rats, and the variability as well.

F1 generation rats

There were no treatment-related clinical signs, gross lesions or changes in body weight, body weight gains, feed consumption or organ weights in the male and female rats at any dosage level tested. Anogenital distance on day 1 or 22 postpartum, nipple eruption on day 12 postpartum and sexual maturation was unaffected in either sex following treatment of the P generation male and female rats with dosages up to and including 500 mg/ kg bw /day.

Based on the results of this study, the NOAEL for parental general systemic toxicity of 3,7-dimethyloct-6-enenitrile is 200 mg/ kg bw /day. The reproductive NOAEL in the P generation rats and the NOAEL for viability and growth of the F1 generation offspring is greater than 500 mg/ kg bw /day. There were no apparent effects on estrous cycling, mating and fertility, reproductive organ weights or natural delivery parameters in the P generation and growth and development (including anogenital distance, nipple eruption or sexual maturation) in the Fl generation rats at the highest dosage level tested (500 mg/ kg bw /day).

Effects on developmental toxicity

Description of key information

OECD 414 (BASF SE, 2016): rat, gavage
- NOAEL (maternal toxicity) = 150 mg/kg bw/d
- NOAEL (prenatal developmental toxicity) = 450 mg/kg bw/d

OECD 414 (BASF SE, 2021): rabbit, gavage
- NOAEL (maternal toxicity) = 80 mg/kg bw/d
- NOAEL (prenatal developmental toxicity) = 250 mg/kg bw/d

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

25 June 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

Aug 1998

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch number of test material: BASF SE, 19000329U0
- Purity: 95.6 area-%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability of the test material during storage: Expiry date: 01 May 2021
The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Homogeneity of the test material: given

OTHER
- Chemical name: 3,7-dimethyloct-6-enenitrile
- Physical state/appearance: Liquid/colorless, clear

Species:

rabbit

Strain:

New Zealand White

Remarks:

Crl:KBL(NZW)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services,
Germany GmbH/ Charles River Laboratories, France
- Housing: Single housing
- Fasting period before study: Not applicable
- Diet (e.g. ad libitum): ad libitum; pelleted maintenance diet rabbit and guinea pig “GLP” (Granovit AG, Kaiseraugst/Switzerland)
- Water (e.g. ad libitum): Drinking water ad libitum
- Acclimation period: At least 5 d before artificial insemination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light (06:00 h - 18:00 h); 12 hours darkness (18:00 h - 06:00 h)

Route of administration:

oral: gavage

Vehicle:

other: 0.5% CMC suspension in deionized water (with 10 mg/ 100 mL Cremophor EL)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- The specific amount of test substance was weighed, topped up with 0.5% CMC suspension in deionized water (with 10 mg/ 100 mL Cremophor EL) in an Erlenmeyer flask and intensely mixed with a magnetic stirrer. Before and during administration, the preparations was kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 0, 0.25, 0.8, 2.50 g/100 mL
- Amount of vehicle (if gavage): 10 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in 0.5% CMC suspension in deionized water (with
10 mg/100 mL Cremophor EL) over a period of a maximum of 7 days at room temperature had been verified prior to the start of the study with the same batch.
Samples of the test substance preparations were sent once (at the beginning of administration) to the analytical laboratory for verification of the concentrations. The samples taken for the concentration control analyses were also used to verify the homogeneity of the samples of the low- and high concentrations each (25 and 250 mg/kg bw/d). Three samples (one from the top, middle and bottom) were taken for each of these preparations from the preparation vessel with a magnetic stirrer running.

Details on mating procedure:

- Impregnation procedure: Artificial insemination (GD 0)
- 0.2 mL of a synthetic hormone (Receptal, Intervet Deutschland GmbH, Unterschleißheim, Germany), which releases LH and FSH from the anterior pituitary lobe, was injected intramuscularly into the female rabbits about 1 h before insemination
- The samples of the ejaculate used for the artificial insemination were derived from male New Zealand White rabbits of the same strain as the females. The male donors were maintained under the same conditions (air conditions, feed and water) as the females used in the study. The day of insemination was referred to as GD 0.

Duration of treatment / exposure:

GD 6-28

Frequency of treatment:

daily

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

80 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In a three-week test study (BASF project No.: 01R0333/03R030), Citronellylnitril was administered to each three non-pregnant New Zealand White rabbits per test group at dose levels of 1000, 600, 300 and 0 (control) mg/kg bw/d. The highest test group was terminated at study day 6 due to severe effects on food consumption and body weights.

In a following maternal toxicity range-finding study (BASF project No.: 20R0333/03R031), Citronellylnitril was administered daily to each five pregnant New Zealand White rabbits per test group from implantation to one day prior to the expected day of parturition (GD 6-28). Dose levels of 300 and 600 mg/kg bw/d were used. At the high-dose level of 600 mg/kg bw/d, the following effects were observed: reduced feces in three out of five animals, no feces in four out of five animals, a pronounced reduction in mean food consumption at GD 6 onwards, a body weight loss, a decreased body weight change. One animal of this test group aborted and was sacrificed on GD 21, the other four animals of this test group were sacrificed premature. At the lower dose of 300 mg/kg bw/d, mean food consumption values were slightly reduced on GD 9-11. One out of five animals showed reduced feces, no feces was noted in two out of five animals. One animal of this test group aborted and was sacrificed on GD 21.

Therefore, based on the above-mentioned range-fining study, the following dose levels were chosen for the present prenatal developmental toxicity study in New Zealand White rabbits: 0, 25, 80 and 250 mg/kg bw/day.

- Rationale for animal assignment: During the acclimatization period the animals were assigned to the different test groups according to a randomization plan (NIJENHUIS and WILF) and on the basis of their body weights.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
- At least once daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity
- A check for moribund and dead animals twice daily from Mondays to Fridays and once daily on Saturdays, Sundays and public holidays (GD 0 to 29)
- During administration period (GD 6-28): all animals were checked daily for abnormal clinical signs before administration as well as within 5 hours after the administration. Abnormalities and changes are documented for each animal

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29

FOOD CONSUMPTION: Yes
- Time schedule for examination: daily during GD 0-29

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Gross-pathological examination
The following weights were determined in all does sacrificed on schedule: adrenal glands, kidneys, liver, spleen
The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution: all gross lesions, adrenal glands, kidneys, liver, spleen

CLINICAL PATHOLOGY
In the morning blood was taken from the ear vein from not-fasted animals without anesthesia. The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence.

Hematology
Parameters examined:
- Leukocyte count (WBC)
- Erythrocyte count (RBC)
- Hemoglobin (HGB)
- Hematocrit (HCT)
- Mean corpuscular volume (MCV)
- Mean corpuscular hemoglobin (MCH)
- Mean corpuscular hemoglobin concentration (MCHC)
- Platelet count (PLT)
- Differential blood count
- Reticulocytes (RETA)

Clinical chemistry
Parameters examined:
- Alanine aminotransferase (ALT)
- Aspartate aminotransferase (AST)
- Alkaline phosphatase (ALP)
- gamma-Glutamyltransferase (GGT)
- Inorganic phosphate (INP)
- Calcium (CA)
- Urea (UREA)
- Creatinine (CREA)
- Glucose (GLUC)
- Total bilirubin (TBIL)
- Total protein (TPROT)
- Albumin (ALB)
- Globulins (GLOB)
- Triglycerides (TRIG)
- Cholesterol (CHOL)

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Other: Site of implantations in the uterus; Gross-pathological examination

Blood sampling:

Additional samples during venipuncture were collected for external research projects beyond the scope of this study and without GLP status. The results of the study were not influenced by this procedure because blood sampling occurred just prior to sacrifice/at necropsy. The last aliquot was taken for these projects, thus blood sampling for the regulatory investigations was not affected. Thus, the sampling procedures did not affect the outcome and compliance of the GLP study. The data from these research projects do not affect the outcome, assessment and compliance of this GLP study.

On GD 29, blood samples were taken from animals from the ear veins. Blood sampling was carried out in a randomized sequence. The list of randomization instructions was compiled with a computer.

From all presumed pregnant animals, 1 mL of blood was collected using EDTA as anticoagu- lant (10 µL of a 10% solution). The samples were centrifuged. The plasma was separated. The blood was sampled and prepared in original Eppendorf tubes. The preparation of the samples was done under cooling. All samples were stored covered with a N2 atmosphere and then stored at –80°C for research.

All these samples were taken for metabolome analysis at BASF Metabolome Solutions GmbH, Tegeler Weg 33, 10589 Berlin, Germany.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter (and the heads of any fetus which revealed severe findings during the external examination, e.g. anophthalmia, microphthalmia or hydrocephalus)

Statistics:

Means and standard deviations were calculated. In addition, the following statistical analyses were carried out:

- DUNNETT’s test: Food consumption, body weight, body weight change, corrected body weight gain, carcass weight, weight of the unopened uterus, weight of the placentas and fetuses, corpora lutea, implantations, pre- and postimplantation losses, resorptions and live fetuses
- FISHER's exact test: Number of pregnant animals at the end of the study, mortality rate (of the does) and number of litters with fetal findings
- WILCOXON test: Proportion of fetuses with findings per litter
- KRUSKAL-WALLIS and WILCOXON-test (two-sided) test: Blood parameters
- KRUSKAL-WALLIS H test (two-sided) and WILCOXON-test (two-sided): Weight parameters

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Only pregnant does were used for the calculations of mean maternal food consumption, body weight and body weight change. Only pregnant does with scheduled sacrifice (GD 29) were used for the calculation of mean gravid uterine weights, mean organ weights, corrected (net) body weight gain and summary of reproduction data.
The following females were excluded from the above-mentioned calculations:
Test group 0 (0 mg/kg bw/d): females Nos. 10, 15 - not pregnant
Test group 1 (25 mg/kg bw/d): female No. 50 - died after gavage error
Test group 2 (80 mg/kg bw/d): females Nos. 59, 64 - not pregnant
Test group 3 (250 mg/kg bw/d): females Nos. 86, 92 - not pregnant

One female, each, of the control and the high-dose groups had blood in bedding before and after treatment: No. 3 (0 mg/kg bw/d) on GD 19 and No. 91 (250 mg/kg bw/d) on GD 25.
Furthermore, low-dose doe No. 50 (25 mg/kg bw/d) showed, pre-terminally, labored respiration after treatment on GD 18 which was caused by a gavage error on this very day.
No defecation was observed in one low-dose, one mid-dose and three high-dose females. Reduced defecation was observed in one control, four low-dose, three mid-dose and three high-dose females (0, 25, 80 and 250 mg/kg bw/d). Incidence and distribution of these findings do not indicate a relationship to the test substance.
There were no further clinical findings in the other does in this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One low-dose female (No. 50 - 25 mg/kg bw/d) died after gavage error on GD 19.
There were no further substance-related or spontaneous mortalities in any of the does in the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant differences were observed for the mean body weights and the average body weight gain of the low-, mid- and high-dose females (25, 80 or 250 mg/kg bw/d) when compared to the control group.
Nevertheless, corresponding to food consumption, the high-dose rabbits gained 35% less weight than the controls during the treatment period (GD 6-28) and also during the study period (GD 0-29, -24%), the latter difference attained statistical significance.

Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were not significantly different between all test groups including controls (0, 25, 80 or 250 mg/kg bw/d).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

The average food consumption of the low-, mid- and high-dose rabbits (25, 80 and 250 mg/kg bw/d) was not significantly different to the concurrent control (0 mg/kg bw/d) throughout the entire study period.
However, the high-dose does consumed less food than the control does on each day of the treatment period, although the difference never gained statistical significance. The overall food consumption of the high-dose group was about 9% below control during the treatment period on GD 6 through 28.
A single food consumption value in the mid-dose group on GD 26-27 which was significantly below control was considered as a spurious finding.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At gestation day 29, red blood cell (RBC) counts, hemoglobin and hematocrit values were decreased
(hematocrit not statistically significantly) in does of test group 3 (250 mg/kg bw/d), whereas absolute reticulocyte counts and mean corpuscular volume (MCV) were significantly increased. These alterations were regarded as treatment-related and adverse.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At gestation day 29, in does of test group 3 (250 mg/kg bw/d) γ-glutamyl transferase (GGT) activities were significantly increased whereas creatinine values were significantly decreased. These alterations were regarded as treatment-related and adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute weights
All mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (=100%), the mean relative weights of the liver were significantly increased in test group 3:
Relative weights Females
Test group 1 (25 mg/kg bw/d): 97%
Test group 2 (80 mg/kg bw/d): 97%
Test group 3 (250 mg/kg bw/d): 107%*
*: p <= 0.05
All other mean relative weight parameters did not show significant differences when compared to the control group 0. The significantly increased relative liver weights in test group 3 animals represent a marginal change, since only the relative weights were significantly increased, the weight increase was very mild and the absolute and relative liver weights lay within the range of historical control values. However, in combination with increased γ-glutamyl transferase (GGT) activities in this test group, a relation to the treatment cannot be ruled out.

Gross pathological findings:

no effects observed

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the different test groups in the values calculated for pre- and post-implantation losses.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no test substance-related and/or biologically relevant differences between the different test groups in the number of resorptions.

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Female rabbits were placed into the study in four cohorts. Each dose group was represented in each cohort. The conception rate was 92% in the control, mid- and high-dose groups (0, 80 and 250 mg/kg bw/d) and 100% in the low-dose group (25 mg/kg bw/d). A sufficient number (approximately 20, but not fewer than 16 females with implantation sites) of pregnant females was available for the purpose of the study.
There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Uterus weights
The mean gravid uterus weight of the rabbits in test group 3 (250 mg/kg bw/d) was significantly reduced (-21% in comparison to the concurrent control). This decrease was coherent with a lower number of corpora lutea and implantation sites and a, consequently, lower number of viable fetuses in this test group (see section 4.2.2.2. Reproduction data). As the potential origin of these findings lies before the start of treatment, an association to the test substance is not assumed.
The mean gravid uterus weight of the rabbits of test groups 1 and 2 (25 or 80 mg/kg bw/d) was not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.

Key result

Dose descriptor:

NOAEL

Effect level:

80 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: mild anemia

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

In test group 3 (250 mg/kg bw/d), the mean number of corpora lutea (8.9* (p ≤ 0.05) vs. 10.6 in control) and as a result of this, mean number of implantation sites (7.8 vs. 9.5 in control) were lower and below their historical control ranges (HCD corp.lut.: mean 10.2 (9.3 - 11.1); HCD impl. sites: mean 9.6 (8.7 - 10.9)). Subsequently, the number of live fetuses (both sexes combined) was also significantly lower (7.0* (p ≤ 0.05) vs. 9.1 in control; HCD viable fetuses
(litter size): mean 9.0 (7.9 - 10.2)). Furthermore, there seems to be a shift towards a lower percentage of female live fetuses (mean% 32.4** (p ≤ 0.01) vs. 50.8 in control) in this group. However, as ovulation, fertilization and formation of corpora lutea take place 6 days before the start of treatment, a relationship of these apparent findings to the test substance cannot be assumed.


All other differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.

One dead fetus was found at C-section of mid-dose doe No. 62 which is a rare finding but may occur spontaneously in this rabbit strain. As there was no evidence for potential developmental toxicity of the test item in this study, this is considered to be an incidental finding.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (25, 80 and 250 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean placental weights in test groups 1, 2 and 3 were not influenced by the test substance and were comparable to the control value.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were recorded for one fetus, each, in test groups 0, 2 and 3 (0, 80 and 250 mg/kg bw/d). In high-dose fetus No. 89-07, the multiple external malformations (short snout, absent nostrils, malpositioned eyes, flattened back of head) were associated with additional visceral and skeletal malformations. However, these findings were isolated events in a single fetus, thus, they are considered to be incidental. No statistically significant differences of overall incidences were noted between the groups

Individual fetal external malformations:
0 (0 mg/kg bw/d), 23-14 F: umbilical hernia
1 (25 mg/kg bw/d): none
2 (80 mg/kg bw/d), 57-02 F: umbilical hernia
3 (250 mg/kg bw/d) 89-07 M a): multiple external malformations
a) fetus with additional visceral and skeletal malformations

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in single fetuses of test groups 1, 2 and 3 (25, 80 and 250 mg/kg bw/d). Two fetuses had additional external and/or soft
tissue malformations. All findings appeared without a dose-response relationship and nearly all of them can be found in the historical control data at comparable incidences, thus, they were assessed as not treatment-related.
In summary, these events added up to a slightly increased affected fetuses per litter-incidence of skeletal malformations in test group 3 which attained statistical significance. However, in all test groups, the overall incidences of skeletal malformations were covered by the historical control range (mean%: 0.8 [0.0 - 2.4]).

Individual fetal skeletal malformations
Test group, Doe No.-Fetus No., Sex: Finding
0 (0 mg/kg bw/d): none
1 (25 mg/kg bw/d), 30-08 F: malpositioned and bipartite sternebra; 49-05 F: additional vertebral arch and corresponding rib, branched rib
2 (80 mg/kg bw/d), 54-12 M b): sternebrae severely fused (bony plate); 69-01 F: sutures fused
3 (250 mg/kg bw/d), 84-01 F: malpositioned and bipartite sternebra; 84-07 F: thoracic hemivertebra; 85-01 F: misshapen lumbar vertebra
89-07 M a)b): multiple skeletal malformations
a) fetus with additional external malformations
b) fetus with additional soft tissue malformations

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Fetal soft tissue malformations
Soft tissue malformations occurred in all test groups including control (0, 25, 80 and 250 mg/kg bw/d). Two fetuses in different test groups had additional external and/or skeletal malformations.
The distribution of the findings about the test groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted. The total incidence of soft tissue malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.
Individual fetal soft tissue malformations
Test group, Doe No.-Fetus No., Sex: Finding
0 (0 mg/kg bw/d), 6-09 M: absent subclavian
1 (25 mg/kg bw/d), 40-03 M: cor triloculare; 49-01 F: malpositioned kidney, short ureter; 49-03 F: absent kidney, absent ureter
2 (80 mg/kg bw/d), 54-12 M b): absent subclavian
3 (250 mg/kg bw/d), 85-03 F: hydronephrosis; 89-07 M a)b): misshapen brain; 97-03 M: absent subclavian
a) fetus with additional external malformations
b) fetus with additional skeletal malformations

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

External examination of the fetuses
- Fetal external variations:
No external variations were recorded.

- Fetal external unclassified observations:
One unclassified observation (‘amniotic fluid discolored’) was recorded in one fetus of test group 2 (80 mg/kg bw/d). This finding is considered not to be related to treatment.


Skeletal examination of the fetuses
- Fetal skeletal variations
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing. The statistically significantly increased incidences of ‘fused sternebra (unchanged cartilage)’ in test group 1 and 3 (affected fetuses/litter, mean%: 0.0/3.1*/1.1/1.9* [p ≤ 0.05]) were not related to the dose and, furthermore, were within the historical control range (HCD: mean% 0.9 [0.0 - 3.1]). Thus, this finding is assessed as not treatment-related.
The overall incidences of skeletal variations were neither statistically significantly different from control nor dose-dependent and, therefore, not considered biologically relevant.

- Fetal skeletal unclassified cartilage observations
Some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the vertebral column, sternum and the ribs. The overall incidences of skeletal unclassified cartilage observations in the substancetreated
groups did not differ significantly from the concurrent control group.
The incidence of ‘bipartite processus xiphoideus’ was statistically significantly increased in test group 2 (affected fetuses/litter, mean%: 2.5/3.5/9.8**/2.4 [p ≤ 0.01]). However, as the value of test group 2 was within the historical control range (mean% 4.8 [0.5 - 22.1]) and there is no dose-response, no association to the treatment is assumed.


Soft tissue examination of the fetuses
- Fetal soft tissue variations
The examinations of the soft tissues revealed malpositioned carotid branches in the test groups 0, 1 and 2. An absent lung lobe (Lobus inferior medialis) was noted in all test groups including the control (0, 25, 80 and 250 mg/kg bw/d). Other variations, such as cystic dilatation of the brain (test group 2), supernumerary branch from aortic arch (test groups 0 and 3), dilated aortic
arch and narrowed pulmonary trunk (test group 1, respectively) and dilated ureter (test groups 1 and 3) occurred in individual fetuses of the different test groups.
No statistically significant or toxicologically relevant differences between the groups were noted, and the overall incidences were within the historical control range.

- Fetal soft tissue unclassified observations
Two unclassified soft tissue observations were recorded. A blood coagulum around urinary bladder was seen in 14 control, six low-dose, five mid-dose and eight high-dose fetuses. This finding can be found in the historical control data at a comparable incidence, therefore, it was neither assessed as treatment-related nor as adverse. Furthermore, a fluid-filled pericardium was recorded in one fetus of test group 0.

Details on embryotoxic / teratogenic effects:

Assessment of all fetal external, soft tissue and skeletal observations
There were noted external (Tab. ID-003), soft tissue (Tabs. ID-007 - ID-009) and skeletal malformations in all test groups (0, 25, 80 or 250 mg/kg bw/d). The distribution of total malformations about the groups was not related to dose.
Three fetuses of the same litter in test group 1 (25 mg/kg bw/d), one fetus in test group 2 (80 mg/kg bw/d) and one fetus in test group 3 (250 mg/kg bw/d) had more than one malformation. Female low-dose fetus No. 49-01 had a malpositioned kidney and a short ureter, while female fetus 49-03 showed an absent kidney and absent ureter; furthermore, for female fetus No. 49-05 an additional vertebral arch with corresponding rib and a branched rib were
recorded. Male mid-dose fetus No. 54-12 had an absent subclavian in combination with severely fused sternebrae (bony plate) during skeletal examination. Finally, male high-dose fetus No. 89-07 was multiple-malformed across the different examination areas. The findings consisted of multiple external malformations, such as short snout, absent nostrils,
malpositioned eyes, flattened back of head, a misshapen brain (small cerebellum, no separation of cerebral hemisphere) and multiple skeletal malformations affecting the skull, cervical and lumbar vertebrae. No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups.
The finding ‘absent subclavian’ which was seen in a multiple malformed mid-dose fetus, occurred also in one individual fetus, each, of test groups 0 and 3. Other malformations, such as umbilical hernia, cor triloculare, hydronephrosis, sutures fused, thoracic hemivertebra, misshapen lumbar vertebra, as well as malpositioned and bipartite sternebra were scattered observations in individual fetuses of all test groups including the control. They all were not dose-related and, except ‘hydronephrosis’ which occurred in one single high-dose fetus, all of
them can be found in the historical control data at comparable frequency. An association of these findings to the treatment is not assumed.

External variations did not occur in any fetus in this study. A spontaneous origin is assumed for the soft tissue variations and the range of skeletal variations which were noted in all test groups including controls. If all different types of
variations are summarized, none of the incidences showed a relation to dosing
and can be found in the historical control data at comparable frequency.

A spontaneous origin is assumed for the unclassified external, unclassified soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of all test groups (0, 25, 80 and 250 mg/kg bw/d). The
distribution and type of these findings do not suggest any relation to treatment.
Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (250 mg/kg bw/d).

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects up to the highest dose tested

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Strain: Crl:WI(Han)
- Age at study initiation: about 10-12 weeks
- Weight at study initiation: 150.9–189.7 g
- Housing: individually in Polycarbonate cages type III with wooden gnawing blocks and dust-free wooden bedding
- Diet: Ground Kliba maintenance diet mouse/rat "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water: tap water ad libitum
- Acclimation period: between the start of the study (beginning of the experimental phase) and the first administration (GD 6)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The specific amount of test substance was weighed, topped up with corn oil in a graduated flask and intensely mixed with a magnetic stirrer until it was completely dissolved.

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory. The following day was designated as “GD 1”.

Duration of treatment / exposure:

GD 6-19

Frequency of treatment:

once daily

Duration of test:

on GD 20, all surviving females were sacrificed

Remarks:

Doses / Concentrations:
50, 150 and 450 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day on working days or once a day on Saturday, Sunday or on public holidays (GD 0-20)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours after administration. If such signs occurred, these animals were examined additionally within 5 hours after administration.

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20

FOOD CONSUMPTION: Yes
- Time schedule for examinations: GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20

HEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule: In the morning of GD 20 blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Pathology: The weights of adrenal glands, liver and spleen were determined in all animals sacrificed. These organs were fixed in 4% neutral-buffered formaldehyde solution. The carcass weights were used to calculate the relative organ weights.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of dead fetuses: Yes

Fetal examinations:

- Weight of each fetus: Yes
- Sex: Yes
- Weight of placentas: Yes
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

Statistics:

- DUNNETT's test: Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass
weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions
of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight
- FISHER's exact test: Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings
- WILCOXON test: Proportions of fetuses with malformations, variations and/or unclassified observations in each litter
- KRUSKAL-WALLIS test: blood parameters, organ weights

Details on maternal toxic effects:

Details on maternal toxic effects:
- MORTALITY:
There were no substance-related or spontaneous mortalities in any females of all test groups.

- CLINICAL SYMPTOMS:
All females of the high-dose group (450 mg/kg bw/d), nearly all (22 out of 25) females of the mid-dose group (150 mg/kg bw/d) and two females of the low-dose group (50 mg/kg bw/d) showed transient salivation during the treatment period (initially observed on GD 6, 0-2h after treatment). Nearly all (21 out of 25) females of the high-dose group (450 mg/kg bw/d) and nine females of the mid-dose group (150 mg/kg bw/d) plough nose-first into bedding (initially observed on GD 13, within 0-2h after treatment). During the 5-hour examination interval (i.e. <2<5h after treatment), no clinical signs or changes of general behavior were detected in any female of all test groups.

- WATER CONSUPTION:
The mid- and high-dose dams consumed 12% / 12% (150 mg/kg bw/d) or 18% / 15% (450 mg/kg bw/d) more water in comparison to the concurrent control group, calculated for the entire treatment period (GD 6-19) / the entire study period (GD 0-20) respectively.
The statistically significantly increased water consumption value of the high-dose females during GD 1-3 (pre-treatment period) was not considered biologically relevant.

- FOOD CONSUMPTION:
The mean food consumption of the high-, mid- and low-dose dams (450, 150 and 50 mg/kg bw/d) was generally comparable to the controls throughout the entire study period.

- BODY WEIGHT:
The mean body weights and the average body weight gain of the high-, mid- and low-dose dams (450, 150 or 50 mg/kg bw/d) were in general comparable to the controls throughout the entire study period.

- CORRECTED (NET) BODY WEIGHT GAIN:
There was no difference of any biological relevance to the corresponding control group for any dose group.

- HEMATOLOGY:
No treatment-related changes among hematological parameters were observed.

- CLINICAL CHEMISTRY:
In dams of test groups 2 and 3 (150 and 450 mg/kg bw/d), cholesterol and triglyceride values were increased. In dams of test group 2 cholesterol mean was within, that one of triglyce-ides was above the historical control range. Because in this test group only one clinical pathology parameter was altered, this change was regarded as treatment-related but not adverse.

- UTERUS WEIGHT:
The mean gravid uterus weights of the animals of test group 1-3 (50, 150 and 450 mg/kg bw/d) were not influenced by the test substance.

- REPRODUCTION DATA:
conception rate:
• 0 mg/kg bw/d: 100%
• 50 mg/kg bw/d: 100%
• 150 mg/kg bw/d: 96%
• 450 mg/kg bw/d: 100%
No test substance related and/or biologically relevant differences between test groups 0, 1, 2 and 3 (0, 50, 150 and 450 mg/kg bw/d) in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and the postimplantation losses, the number of resorptions and viable fetuses.

- PATHOLOGY:
A dose related increase of the absolute and relative organ weights of the liver was recorded for all test groups
absolute / relative
• 50 mg/kg bw/d: 107% / 106%
• 150 mg/kg bw/d: 110% / 110%
• 450 mg/kg bw/d: 119% / 121%
All other mean relative weight parameters showed no significant differences when compared to the control group 0.
No gross lesions were noted in any female of all test groups (0, 50, 150 or 450 mg/kg bw/d).

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: increased liver weights and corresponding clinical-pathological changes (increased triglyceride and cholesterol values)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
- SEX DISTRIBUTION:
comparable to the concurrent control fetuses

- WEIGHT OF PLACENTAE:
comparable to the concurrent control group

- WEIGHT OF FETUSES:
no test substance related effects

- FETAL EXTERNAL MALFORMATIONS:
One external malformation was detected in test group 3 (450 mg/kg bw/d), i.e. gastroschisis. The total incidence of external malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.

- FETAL EXTERNAL VARIATIONS:
One external variation was detected in one fetus of test group 3 (450 mg/kg bw/d), i.e. limb hyperextension. The total incidence of external variations in treated animals did not differ significantly from the control group and was comparable to the historical control data.

- FETAL EXTERNAL UNCLASSIFIED OBSERVATIONS:
One unclassified external observation, i.e. placentae fused, was recorded in one fetus of the high-dose group (450 mg/kg bw/d). This finding was not considered biologically relevant.

- FETAL SOFT TISSUE MALFORMATIONS:
no external malformations were recorded in the fetuses of the treated groups

- FETAL SOFT TISSUE VARIATIONS:
Four soft tissue variations were detected in all test groups including the control (0, 50, 150 or 450 mg/kg bw/d), i.e. short innominate, carotid: supernumerary branch, dilated renal pelvis and dilated ureter. The incidences of these variations were neither statistically significantly different from control nor dose-dependent and therefore, not considered biologically relevant.

- FETAL SOFT TISSUE UNCLASSIFIED OBSERVATIONS:
no unclassified soft tissue observations were recorded

- FETAL SKELETAL MALFORMATIONS:
Three test group 3 (450 mg/kg bw/d) fetuses from 3 different litters had skeletal malformations affecting the vertebral column and the sternum. The number of these malformations adds up to a statistically significantly higher value for the total skeletal malformation rate in the high-dose group. However, the respective value is clearly inside the historical control range (HCD: 0.8% [0.0 - 3.0]).

- FETAL SKELETAL VARIATIONS:
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared in the majority of cases without a relation to dosing. The overall incidences of skeletal variations were comparable to the concurrent and historical controls.

- FETAL SKELETAL UNCLASSIFIED CARTILAGE OBSERVATIONS:
Some isolated cartilage findings without impact on the respective bony structures, which were designated as unclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum and, in most cases, did not show any relation to dosing. The incidence of ‘notched cartilage between basisphenoid and basioccipital’ was statis-tically significantly increased in test group 3 (5.3% affected fetuses per litter) but was well inside the historical control range (HCD: 2.4% [0.0 - 10.0]). The overall incidences of skeletal unclassified cartilage observations in the substance-treated groups did not differ significantly from the concurrent control group.

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects observed up to the highest dose tested

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

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

Data on developmental toxicity for Citronellylnitril is available in two species (rat and rabbit).

Citronellylnitril was tested for its prenatal developmental toxicity in Wistar rats according to OECD 414 and GLP (BASF SE, 2016). The test substance was administered as an oily solution to groups of 25 time-mated female Wistar rats by gavage at doses of 50, 150 and 450 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 19. The control group, consisting of 25 females, was dosed with the vehicle (corn oil) in parallel. A standard dose volume of 4 mL/kg body weight was used for each test group. On GD 20, blood samples were obtained from all females by retrobulbar venous puncture following isoflurane anesthesia. After blood sampling all females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology. For each dam, corpora lutea were counted and number and distribution of implantation sites were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, fetuses were examined for soft tissue findings and the remaining fetuses for skeletal findings.

Under the conditions of this prenatal developmental toxicity study, the oral administration of Citronellylnitril to pregnant Wistar rats caused evidence of maternal toxicity at 450 mg/kg bw/d, namely increased liver weights (absolute +19% and relative +21%) and corresponding clinical-pathological changes (increased triglyceride and cholesterol values). In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 150 mg/kg bw/d. There were no toxicologically relevant adverse fetal findings evident. Thus, the no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 450 mg/kg bw/d.

 

 

Citronellylnitril was tested for its prenatal developmental toxicity in New Zealand White rabbits (BASF SE, 2021). The test substance was administered as an aqueous suspension to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 25, 80 and 250 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 28. The vehicle control group, consisting of 25 females, was dosed with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in deionized water [with 10 mg/100 mL Cremophor EL]) in parallel.

A standard dose volume of 10 mL/kg body weight was used for each test group.

At terminal sacrifice on GD 29, 23-24 females per group had implantation sites.

Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 29, blood samples were obtained from all females from the ear veins. After blood sampling, all surviving females were sacrificed and assessed by gross pathology (including weight determinations of the adrenal glands, kidneys, liver, spleen, unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings.

The following test substance-related adverse effects/findings were noted at test group 3 (250 mg/kg bw/d):

• Lower food consumption (9% below control) during the treatment period,


• Reduced body weight gain during the treatment period (GD 6-28, 35% below controls), and during the entire study period (GD 0-29, 24% below controls),

• Decreased red blood cell (RBC) counts, hemoglobin and hematocrit values,


• Increased absolute reticulocyte counts and mean corpuscular volume (MCV),


• Increased γ-glutamyl transferase (GGT) activities,


• Decreased creatinine values.


• Increased relative liver weight.


• No test substance-related adverse effects on gestational parameters or fetuses.

No test substance-related adverse effects on does, gestational parameters or fetuses were noted at test group 2 (80 mg/kg bw/d) and test group 1 (25 mg/kg bw/d).

Under the conditions of this prenatal developmental toxicity study, the oral administration of Citronellylnitril to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) caused evidence of beginning maternal toxicity at a dose of 250 mg/kg bw/d, such as slightly but consistently decreased food consumption and weight gain as well as mild anemia.

In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 80 mg/kg bw/d.

The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 250 mg/kg bw/d, the highest dose tested.

The test substance is not teratogenic in rabbits at the tested dose levels.

Justification for classification or non-classification

The present data on reproductive toxicity do not fulfill the criteria laid down in regulation (EU) 1272/2008, and therefore, a non-classification is warranted.