Citral

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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)

Remarks:

BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

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 : 2 weeks; according to ECHA TP decision TPE-D-2114460918-36-01/F
F1 animals of Cohort 1B are mated to produce the F2 generation and, thus, the premating exposure duration will be ten weeks for these animals. Consequently the fertility parameters will be covered allowing an evaluation of the full spectrum of effects on fertility in these animals. Thus, shorter premating exposure duration for parental (P) animals may be considered.
- Basis for dose level selection : Based on a range finding study
- Inclusion of extension of Cohort 1B: yes; according to ECHA TP decision TPE-D-2114460918-36-01/F
- Termination time for F2 : terminal sacrifice of the F2 weanlings.
- Exclusion of developmental neurotoxicity Cohorts 2A and 2B : according to ECHA TP decision TPE-D-2114460918-36-01/F
- Exclusion of developmental immunotoxicity Cohort 3 : according to ECHA TP decision TPE-D-2114460918-36-01/F
- Route of administration : oral (gavage): according to ECHA TP decision TPE-D-2114460918-36-01/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.

Test material

Test material information

Constituent 1

Reference substance name:

Citral

EC Number:

226-394-6

EC Name:

Citral

Cas Number:

5392-40-5

Molecular formula:

C10H16O

IUPAC Name:

3,7-dimethylocta-2,6-dienal

Test animals

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, Germany GmbH,
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 14 weeks old at the beginning of treatment (P)
- 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: up to 5 animals per sex and cage
From delivery to randomization, during overnight matings (male and female mating partners were housed together), gestation, lactation and females after weaning the animals were housed individually. Dams and their litters were housed together until PND 21.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: approx. 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 02 Dec. 2019 (TS administration) To: 09 Jun. 2020 (sacrifice of F1 cohort 1B animals)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC suspension in drinking water (with 5 mg/ 100 mL Tween 80)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0.25, 0.8, 2.5 g/ml in low, mid and high dose, respectively
- Amount of vehicle (if gavage): 10 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
- Any other deviations from standard protocol:
Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group except for one case in the high dose test group F1 (Co1B), which was paired with 2 females.

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 0.5% CMC suspension in drinking water (with 0.5 mg/ 100 mL Tween 80) in a refrigerator over a period of 7 days at room temperature were verified prior to the start of the study in a similar batch.

At the beginning (during premating) of the administration 3 samples each were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time point additionally one sample from the mid concentration was taken for concentration control analysis. Once during the middle and once during the end of the administration each 1 sample was taken from the low, mid and high concentration for a concentration control analysis.

Duration of treatment / exposure:

F0 males: 2 weeks (premating) + 2 weeks (mating) + max. 6 weeks (post-mating)
F0 females: 2 weeks (premating) + 2 weeks (mating) + approx. 6 weeks (pregnancy + lactation)
F1 males (Co 1B): 10 weeks (post-weaning/premating) + 2 weeks (mating) + 4 weeks (post-mating)
F1 males (Co 1B): 10 weeks (post-weaning/premating) + 2 weeks (mating) + approx. 6 weeks (pregnancy + lactation)
F1 animals (Co 1A): post weaning until an approx. age of 13 weeks

Frequency of treatment:

once daily
Females in labor were not treated.

Details on study schedule:

F0 parental animals:
After a minimum of 2 weeks after the beginning of treatment, males and females from the same dose group were mated. 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 Cohorts 1A and 1B. During 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, to be placed into cohorts. 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.

F1 parental animals (Cohort 1B):
After weaning, 25 male and 25 female F1 pups per test group became F1 generation parental animals. These animals were chosen by lot and each litter was taken into account as far as technically feasible. If fewer than 25 litters were available in a group or if one sex was missing in a litter, more animals were taken from the other litters of the respective test group to obtain the required number of animals to be paired. All selected animals were treated with the test substance at the same dose level as their parents, from post-weaning through adulthood. After a minimum of 10 weeks after assignment of the F1 generation parental animals, the males and females were mated, overnight. The partners were randomly assigned, mating of siblings was avoided. The females were allowed to deliver and rear their pups (F2 generation pups) until PND 4 (standardization) or PND 21. Before weaning of the F2 pups the F1 generation parental male animals were sacrificed. The F1 generation parental females were sacrificed, shortly after the F2 generation pups had been weaned.

Standardization of litters (F1 and F2 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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

80 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

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: based on range-finding study (see "Any other information on results incl. tables")
- Fasting period before blood sampling for clinical biochemistry: yes, for about 16-20 hours

Positive control:

not applicable

Examinations

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.
• F0 and F1 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 and F1 parental animals and F1 rearing animals, with the following exceptions:
• not determined after the 2nd premating week (male F0 animals) and after the 10th premating week (male F1 animals).
• determined with evidence of sperm of the F0 and F1 females for GD 0-7, 7-14 and 14-20.
• detemined for the F0 and F1 females, which gave birth to a litter 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: Yes
- Time schedule for examinations: once a week for the male and female F0 and F1 parental animals and F1 rearing animals, with the following exceptions:
• not determined after the 10th premating week (male F1 parental animals) and during the mating period (male and female F0 parental animals)
• determined with evidence of sperm of the F0 and F1 females for GD 0-1, 3-4, 7-8, 10-11, 14-15, 17-18 and 19-20.
• determined for F0 and F1 females, which gave birth to a litter for PND 1-2, 4-5, 7-8, 10-11, 14-15, 17-18 and 20-21.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period and in the females after weaning.


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):

evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 2 weeks and for all F1 female parental rats (= cohort 1B) for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.
In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75.
At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female and cohort 1A and 1B 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.

- 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.

- 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 F1 and F2 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.

- Vaginal opening
All female F1 pups selected to become the F1 parental generation females (cohort 1B) and F1 rearing animals (cohort 1A) 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 parental generation females (cohort 1B) and F1 rearing animals (chort 1A) 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)

Hormone analysis (F1; 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.
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.


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. Parathyriod glands
26. Pituitary gland
27. Prostate
28. Peyer’s patches
29. Rectum
30. Sciatic nerve
31. Seminal vesicles
32. Skeletal muscle
33. Spinal cord (cervical, thoracic, lumbar)
34. Spleen
35. Stomach (forestomach and glandular stomach)
36. Testis, left
37. Thymus
38. Thyroid glands
39. Trachea
40. Urinary bladder
41. Uterus
42. Vagina
43. Vas deferens

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).

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 control and high dose test groups 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 about 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)
On PND 4, as a result of standardization, all surplus F1 pups were sacrificed by decapitation under isoflurane anesthesia and blood was sampled for determination of serum thyroid hormone concentrations. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.

On PND 22, the surplus F1 generation pups that were not used for the formation of the cohorts or any investigations were sacrificed under isoflurane anesthesia with CO2 and were examined for pathology. The selected pups for hormone analyses were sacrificed by decapitation under isoflurane anesthesia blood was sampled for thyroid hormone analyses. All animals were necropsied and assessed by gross pathology with special emphasis on the reproductive organs.

- Organ weights (F1 pups; PND 22)
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)


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; special attention being given to the reproductive organs.

- 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. Parathyriod glands
26. Pituitary gland
27. Prostate
28. Peyer’s patches
29. Rectum
30. Sciatic nerve
31. Seminal vesicles
32. Skeletal muscle
33. Spinal cord (cervical, thoracic, lumbar)
34. Spleen
35. Stomach (forestomach and glandular stomach)
36. Testis, left
37. Thymus
38. Thyroid glands
39. Trachea
40. Urinary bladder
41. Uterus
42. Vagina
43. Vas deferens


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. Seminal vesicles including coagulating gland (fixed)
10. Spleen
11. Testes
12. Thyroid glands
13. 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. Adrenal glands
2. Cervix uteri
3. Coagulating glands
4. Epididymides
5. Ovaries
6. Oviducts
7. Prostate
8. Pituitary gland
9. Seminal vesicles
10. Testes
11. Uterus
12. Vagina

The uteri of all cohabited female cohort 1B 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).


PATHOLOGY (F2 pups)
After a similar standardization on PND 4, the surplus F2 pups were sacrificed under isoflurane anesthesia with CO2.
On PND 21, all F2 generation pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.

- Pup organ weights (F2 pups)
After the scheduled sacrifice, the brain, spleen and thymus of 1 pup/sex per F2 litter were weighed. Normally, the first male and the first female pups/litter were taken for these determinations. The corresponding in-life pup weights determined on PND 21 were used to calculate the relative organ weights.

Statistics:

- DUNNETT test (two-sided): Water consumption (parental and rearing animals), food consumption (parental and rearing animals), body weight and body weight change (parental animals, rearing animals and pups; for the pup weights, the litter means were used), gestation days, 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 mated, females pregnant, females delivering, females with liveborn pups, females with stillborn pups, females with all stillborn pups
- WILCOXON-test (one-sided): 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+) with BONFERRONI-HOLM adjustment: Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal Loss, nipple development
- WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment: Implantation sites, pups delivered, pups liveborn, live pups day x, viability index, lactation index
- WILCOXON-test (one-sided-): DOFC
- WILCOXON test (two-sided): % live male day x, % live female day x
- KRUSKAL-WALLIS test (two-sided) + WILCOXON-test (two-sided): Number of cycles and Cycle Length, pup organ weights (absolute and relative), 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
Lactation index (%) = number of live pups on day 21 after birth / number of live pups on day 4* after birth x 100

*before standardization of litters (i.e. before culling)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males / females (exept gestation/lactation period):
All high-dose male and nearly all high-dose female animals as well as some mid-dose male animals showed salivation at least at one occasion during the study. Salivation occurred immediately after dosing (up to 2 hours post dosing) and wore off afterwards.

Females during gestation of F1 litters:
Nearly all high-dose female animals and two mid-dose female animals showed salivation at least at one occasion during the gestation period. Salivation occurred immediately after dosing (up to 2 hours post dosing) and wore off afterwards.

Females/offspring during lactation of F1 litters:
All high-dose female animals and one mid-dose female animal showed salivation at least at one occasion during the lactation period. Salivation occurred immediately after dosing (up to 2 hours post dosing) and wore off afterwards.

Other observations observed were not considered to be associated with the test compound administration.

Detailed clinical observation (DCO):
Four high-dose female animals showed salivation on DCO day 57.

No other 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.

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.

1 F0 female animal (mid dose group) was found dead on PND 12. Most likely a gavage error was the cause of this preterminal death.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights:
- statistically significantly above the concurrent control values in high-dose F0 females on PND 18 (about 4%), while comparable to the control during the remaining study.

Mean body weights of the low-and mid-dose F0 female animals were comparable to the concurrent control throughout the entire study.
Mean body weights of all test substance-treated F0 male animals were comparable to the concurrent control throughout the entire study.

Body weight change:
- statistically significantly below concurrent control in high-dose F0 males during study days 0 – 7 (about 39%), and significantly above concurrent control during study days 13 - 21 (6.3 g** [**:p<=0.01] vs. 0.1 g in control).
- statistically significantly above the concurrent control values in mid-dose F0 males during study days 13 - 21 (4.7 g* [*:p<=0.05] vs. 0.1 g in control). It was unchanged during the remaining study.
- statistically significantly above the concurrent control values in high-dose F0 females during GD 7 - 14 and 0 - 20 (about 11% and 8%, respectively), and significantly below the concurrent control during PND 18 – 21 (about 58%). It was unchanged during the remaining study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- statistically significantly above the concurrent control in high-dose F0 females during GD 7 - 20 and 0 - 20 (up to 6% and 5%, respectively).

Food consumption of the low- and mid-dose females during the gestation period and of all test substance-treated females during the premating and lactation period was comparable to the concurrent control.
Food consumption of all test substance-treated male animals was comparable to the concurrent control values throughout the entire study.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

- statistically significantly above the concurrent control in high-dose females during the premating period and on GD 3 - 20 (up to 24% and 37%, respectively).
- statistically significantly above the concurrent control values in low- and mid-dose females during GD 17 - 20 (up to 19% and 22%, respectively).

Water consumption of the low- and mid-dose females during the premating period and of all test substance-treated females during the lactation period was comparable to the concurrent control.
Water consumption of all male animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.

- Relative, large unstained cell (LUC) counts were significantly decreased in high dose males at the end of the administration period.
- Absolute LUC counts were significantly increased in high dose females.
Because both cell counts were within historical control ranges, these alterations were regarded as incidental and not treatment related.
- Mean corpuscular hemoglobin concentration (MCHC) was significantly decreased in low and mid dose group males.
This alteration was not dose dependent and therefore, it was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.

- Urea values were significantly increased in mid dose females at the end of the administration period.
- Potassium values were significantly decreased in low dose females.
Since the changes were not dose dependent, they were regarded as incidental and not treatment-related.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormones
In test substance treated animals no treatment-related alterations of T4 and TSH levels were observed.

- T4 values were significantly decreased in high dose females, but the mean value was within the historical control range.
TSH mean value among these individuals was also within the historical control range. Therefore, this change was regarded as incidental and not treatment related.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- incidences of transitional epithelial cells in the urine sediment were significantly increased in high dose males.
This finding was not observed in females. In this age of male rats, this isolated observation is often due to alpha 2u-Globulinuria which is regarded as a species-specific effect in rats without human relevance.
Overall, no treatment-related, adverse changes among urinalysis parameters were observed.

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):

The forestomach was the target organ in male and female mid and high dose group animals . There was a variety of findings ranging from erosions/ulcer through squamous cell hyperplasia (diffuse of focal) and hyperkeratosis (diffuse or focal). These findings were considered as treatment-related and adverse.

- Forestomach (males): erosions/ulcer in 1 mid and 1 high dose animal
- Forestomach (females): erosions/ulcer in 1 high dose animal
- Forestomach (males): squamous cell hyperplasia (diffuse / focal) in 1/9 mid and 9/8 high dose animals
- Forestomach (females): squamous cell hyperplasia (diffuse / focal) in 2/4 mid and 10/4 high dose animals
- Forestomach (males): hyperkeratosis (diffuse or focal) in 1/9 mid and 9/8 high dose animals
- Forestomach (females): hyperkeratosis (diffuse or focal) in 2/4 mid and 10/4 high dose animals


In the heart of high dose males, a slight increase in number of necrosis/fibrosis myocardial was observed (8 animals vs. 3 animals in controls). It is a well-known spontaneous finding especially in male rats, increasing in severity with age. As the findings in the high dose males were only minimal, they were regarded to be spontaneous and incidental.

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 control group female, which was not pregnant did not show relevant histopathological findings consistent with impaired fertility. The male mating partner had hypoplasia of the testes and hypoplasia and aspermia of the epididymides. This was regarded to be the cause of not having offspring in this mating pair.

Decedents
The mid dose female, that was found dead, showed an inflammation of the pleura. Most likely the pleuritis was caused by a gavage error.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

- Mean estrous cycel duration was 3.9, 3.9, 4.0 and 3.9 days in control, low, mid and high dose groups, respectively.

Estrous cycle data, generated during the last 2 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 similar.

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):

Males:
- Male mating index was 100% in all test groups. Copulation was confirmed for all F0 parental males, which were placed with females to generate F1 pups.
- 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. Fertility was proven for most parental males within the scheduled mating interval for F1 litter. One control male did not generate F1 pups. Histopathology revealed hypoplasia of the testes and hypoplasia and aspermia of the epididymides as reason for the infertility.

Females:
- Female mating index was 100% in all test groups.
- Mean duration until copulation was detected (GD 0) varied between 2.2 and 2.7 days without any relation to dosing.
- All female rats delivered pups except: 1 female in control group (mated with male animal described above) did not become pregnant.
- Female fertility index ranged between 96% and 100%. The apparently infertile female rat did not show histopathological findings that could explain infertility.
- Mean duration of gestation was similar in all test groups (i.e. between 22.0 and 22.2 days).
- Gestation index was 100% in all test groups.
- Implantation was not affected by the treatment. Mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (13.4 / 12.6 / 14.0 and 14.2 implants/dam in control, low mid and high dose groups, respectively)
- Postimplantation loss did not show any statistically significant differences between the groups (5.0 / 9.9 / 6.0 and 7.0 mean% in control, low mid and high dose groups, respectively).
- Mean number of F1 pups delivered per dam remained unaffected (12.8 / 11.2 / 13.2 and 13.2 pups/dam in control, low mid and high dose groups, respectively).
- Live birth indices were 99.3% / 99.6% / 99.4% and 99.7% in control, low mid and high dose groups, respectively
- Number of stillborn pups was not significantly different between the test groups.

Thus, no indications for test substance-induced intrauterine embryo-/fetolethality. The rate of liveborn pups was also not affected by the test substance. The test substance did not adversely affect reproduction and delivery of the F0 generation parental animals. The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Remarks:

fertility/reproductive performance

Effect level:

250 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no adverse TS related effects observed

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1B

Males / females (exept gestation/lactation period):
All high-dose male and nearly all female animals and some mid-dose male and one female animal showed salivation during the study. Salivation occurred immediately after dosing (up to 2 hours post dosing) and wore off afterwards.

Females during gestation of F2 litters:
Nearly all high-dose female animals and two mid-dose female animals showed salivation during the gestation period. Salivation occurred immediately after dosing (up to 2 hours post dosing) and wore off afterwards.

Females/offspring during lactation of F2 litters:
All high-dose female animals showed salivation during the lactation period. Salivation occurred immediately after dosing (up to 2 hours post dosing) and wore off afterwards.

No further 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.

Detailed clinical observations (DCO):
One control female was found dead on DCO day 79. No further 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.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

F1 rearing animals, Cohort 1B
There were no test substance-related mortalities in any of the groups.
Two rearing males in the high dose group died three and two days before commencement of cohort 1B premating period, due to gavage error and accidental death, respectively. No replacement of these animals was made.
One female animal of the high dose group was found dead on premating day 7 and one control female was found dead on GD 2. The most likely cause of death of these animals was a gavage error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights
Females:
- statistically significantly above the concurrent control in high-dose females on study day 63, during GD 14 - 20, PND 0 - 4 and 10 - 21 (up to 5%, 7%, 8% and 7% above control, respectively)
Males:
- statistically significantly above the concurrent control in low-dose males during study days 28 - 63 and 84 - 91 (up to 6%, respectively).

Mean body weights of all other test substance-treated groups were comparable to the concurrent control throughout the entire study.

Mean body weight change
Females:
- statistically significantly above the concurrent control in high-dose females during premating days 21 - 28, 56 - 63, 0 - 70 and GD 7 - 14 (about 17%, 40%, 7% and 20%, respectively)
- statistically significantly below the concurrent control in high-dose females during PND 4 – 7 (about 66%)
Males:
- statistically significantly above the concurrent control in high-dose males during study days 7 - 14, 21 - 28 and 49 - 56 (up to 24%)
- statistically significantly below the concurrent control in high-dose males during study days 91 – 98 (about 31%)
- statistically significantly above the concurrent control in mid-dose males during study days 7 - 14 and, 21 - 35 (up to 14%)
- statistically significantly above the concurrent control in low-dose males during study days 7 - 14 and 21 - 28 (up to 9%)

Mean body weight change of all other test substance-treated groups animals was comparable to the concurrent control throughout the entire study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1B

- statistically significantly above the concurrent controls in high-dose females during GD 7 - 20 and 0 - 20 (up to 9%).
- statistically significantly above the concurrent controls in high-dose males during study days 28 - 35, 49 - 56, 56 - 63 and 0 - 70 (up to 9%).
- statistically significantly above the concurrent controls in low- and mid-dose males during study days 28 - 35 (about 7% and 6%, respectively).

No significant changes of food consumption were noted during the remaining study. Food consumption of the low- and mid-dose females was comparable to the concurrent control throughout the entire study.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1B

- statistically significantly above the concurrent control in high-dose females during premating days 42 - 52 and GD 7 - 20 (up to 21% and 31%, respectively).
- statistically significantly above the concurrent control in low-dose females during GD 19 - 20 (about 21%).

Water consumption of test substance-treated female animals was comparable to the concurrent control values in other dose groups or other study periods.
Water consumption of all test substance-treated male animals was comparable to the concurrent control values throughout the entire study.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1B

Absolute organ weights
- Liver (males): 108%* / 106% / 119%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 98% / 103% / 108%* versus ctrl in low, mid and high dose groups, respectively
- Adrenal glands (males): 113%** / 106% / 102% versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

Relative organ weights
- Liver (males): 103% / 104% / 117%** versus ctrl in low, mid and high dose groups, respectively
- Prostate (males): 88%** / 93% / 90%* versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

The increase of liver weight in high dose males and females was regarded to be treatment-related, but not as adverse.
The weight changes in adrenal glands and prostate were considered as incidental due to a missing dose-response relationship and only absolute or relative weights were altered.
All other mean weight parameters did not show significant differences when compared to the control group.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1B

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.

Fertility
The control group female, which was not pregnant had effusion in the thoracic cavity, dark red discoloration of the lungs, and adhesion of the pericard and the lungs. The male mating partner did not show relevant gross lesions.

Decedents
One high dose male and the control group female showed effusion in the thoracic cavity and foamy content or red discoloration of the lungs. This was most likely caused by a gavage error. The high dose female animal revealed no macroscopic findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

F1 rearing animals, Cohort 1B
Fertility
The control group female, which was not pregnant did not show microscopic findings in the sex organs and also the male mating partner did not show microscopic findings which could explain not having offspring.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

F1 rearing animals, Cohort 1B
- Mean estrous cycle duration of 4.2, 4.1, 4.1 and 4.0 days in control, low, mid and high dose animals.

Estrous cycle data, generated during 3 weeks prior to mating to produce the F2 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration was similar.

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
- Male mating index was 100% in all test groups. Copulation was confirmed for all F1 parental males, which were placed with females to generate F2 pups.
- 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. Fertility was proven for most F1 parental males within the scheduled mating interval for F2 litter. One control male did not generate F2 pups. No histopathological findings that could explain infertility were observed.

Females:
- Female mating index was 100% in all test groups.
- Mean duration until copulation was detected (GD 0) varied between 2.3 and 2.7 days without any relation to dosing.
- All female rats delivered pups except: 1 female in control group (mated with male animal described above) did not become pregnant.
- Female fertility index ranged between 96% and 100%. The apparently infertile female rat did not show histopathological findings that could explain infertility.
- Mean duration of gestation was similar in all test groups (i.e. between 21,8 and 21.9 days).
- Gestation index was 100% in all test groups.
- Implantation was not affected by the treatment. Mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.6, 12.9, 13.2 and 13.0 implants/dam in control, low mid and high dose groups, respectively)
- Postimplantation loss did not show any statistically significant differences between the groups (6.2, 4.5, 5.0 and 7.3 mean% in control, low mid and high dose groups, respectively).
- Mean number of F2 pups delivered per dam remained unaffected (11.9, 12.3, 12.5 and 12.2 pups/dam in control, low mid and high dose groups, respectively).
- Live birth indices were 99.6%, 100%, 99.0% and 99.0% in control, low mid and high dose groups, respectively
- Number of stillborn pups was not significantly different between the test groups.

Thus, no indications for test substance-induced intrauterine embryo-/fetolethality. The rate of liveborn pups was also not affected by the test substance. The test substance did not adversely affect reproduction and delivery of the F1 generation parental animals. The mean number of delivered F2 pups per dam and the rates of liveborn and stillborn F2 pups were evenly distributed about the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F1 generation pups/litters
No test substance related adverse clinical signs were observed in the different test groups.
One high-dose female pup was not properly nursed during PND 7 - 8. This observation was not considered to be associated with the test compound.

F1 rearing animals, Cohort 1A
All high-dose male and female animals showed salivation during the entire study. Salivation occurred immediately after dosing (up to 2 hours post dosing) and wore off afterwards.
No further 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.

Detailed clinical observation (DCO):
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the animals in any of the groups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1 generation pups/litters
- Viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99.7% / 98.9% / 99.2% and 98.6% in control, low, mid and high dose group, respectively.
- Lactation index indicating pup survival on PND 4 - 21 was 100% / 99.2% / 94.8% and 98.4% in control, low, mid and high dose group, respectively.
There was a statistically significantly lower mean number of live F1 pups in low dose group animals during PND 7 – 21. One entire mid-dose litter was killed because their mother died after gavage error. Both findings were considered to be spontaneous in nature and not test substance-related. Thus, the test substance did not influence pup survival in any of the treated groups.

F1 rearing animals, Cohort 1A
There were no test substance-related or spontaneous mortalities in any of the groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation pups/litters
Mean body weights and body weight change in all test substance-treated groups were comparable to the concurrent control values throughout the entire study.
A statistically significantly higher body weight change in the low-dose pups (PND 1 - 4 and 1 - 21) was considered to be spontaneous in nature and not treatment-related.

F1 rearing animals, Cohort 1A
Mean body weights and body weight change of all test substance-treated animals were comparable to the concurrent control values throughout the entire study.
The statistically significantly decreased mean body weights in the mid-dose females (on study day 14, 28 and 56), as well as the statistically significantly decreased body weight change in the mid-dose females (during study days 21 - 28) were considered to be spontaneous in nature and not as treatment-related.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1A
Food consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study. A statistically significantly decreased food consumption in the low-dose males (during study days 42 - 49) was considered to be spontaneous in nature and not as treatment-related.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

F1 rearing animals, Cohort 1A
Water consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control throughout the entire study.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 (cohort 1A) animals
No treatment-related changes among hematological parameters were observed.

- prothrombin time (i.e. Hepatoquick’s test, HQT) was significantly reduced in mid dose males.
Since his change was not dose dependent, it was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

F1 (cohort 1A) animals

- Total bilirubin values were significantly decreased in high dose males.
Without any signs of anemia, this finding was most probably due to an increased conjugation rate of bilirubin coupled with an accelerated excretion via the bile because of liver enzyme induction. This effect was regarded as treatment related but adaptive rather than adverse.
Thus, no treatment-related, adverse changes among clinical chemistry parameters were observed.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

F1 (cohort 1A) animals
No treatment-related, adverse changes among urinalysis parameters were observed.

- significantly higher incidences of transitional epithelial cells and granular and epithelial cell casts in the urine sediment in mid and high dose males.
- urine pH values were significantly decreased in high dose males
No corresponding findings were observed in females. In this age of the F1A male rats, these isolated observations are often due to alpha2µ-Globulinuria which is regarded as a species-specific effect in rats without human relevance.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation pups/litters
Sex ratio
No substantial differences between the control and the test substance-treated groups on the day of birth and on PND 21; slight differences were regarded to be spontaneous in nature.

Vaginal opening
- First day of vaginal opening was PND 27
- Last day of vaginal opening was PND 37.
- Mean number of days to reach the criterion was 30.9, 31.3, 32.2** (**:p<=0.01) and 31.3 days in the control, low mid and high dose groups, respectively.
- The mean body weight on the day of vaginal opening was 99.1 g, 101.0 g, 104.7* g (*:p<=0.05) and 99.5 g in the control, low mid and high dose groups, respectively.
The apparent delay in the mid-dose group is considered a spontaneous finding, as it is not dose-related and the average age at puberty is close to the mean of the historical control range (29.5 - 38.8 days).

Preputial separation
- First day of peputial separation was PND 39
- Last day of peputial separation was PND 50
- Mean number of days to reach the criterion was 41.7, 42.2, 42.7* and 42.8* days in the control, low mid and high dose groups, respectively.
- The mean body weight on the day of preputial separation was 179.4 g, 186.4 g, 186.1 g and 189.2* g in the control, low mid and high dose groups, respectively.
The apparent delay in the mid- and high-dose groups is considered as a spontaneous finding, as its magnitude is not dose-related and the average age at puberty is, for both groups, close to the mean of the historical control range (40.1 - 45.2 days).

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation pups/litters
The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.
The statistically significantly increased anogenital distance and anogenital index in the low-dose male pups was considered to be spontaneous in nature and not treatment-related, as it was not dose-related and inside the historical control range.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

F1 generation pups/litters
- The percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13.
- On PND 20 no nipples/areolae were detected in any male pup of all test groups.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Surplus F1 generation pups on PND 22

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


F1 rearing animals, Cohort 1A

Absolute organ weights
- Kidneys (males): 103% / 104% / 115%** versus ctrl in low, mid and high dose groups, respectively
- Liver (males): 99% / 104% / 115%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 100% / 96% / 112%** versus ctrl in low, mid and high dose groups, respectively
- Cauda epididymis (males): 104% / 101% / 114%** versus ctrl in low, mid and high dose groups, respectively
- Prostate (males): 95% / 91%* / 105% versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

Relative organ weights
- Kidneys (males): 104% / 103% / 117%** versus ctrl in low, mid and high dose groups, respectively
- Liver (males): 101% / 104% / 116%** versus ctrl in low, mid and high dose groups, respectively
- Liver (females): 101% / 101% / 111%** versus ctrl in low, mid and high dose groups, respectively
- Cauda epididymis (males): 105% / 100% / 115%** versus ctrl in low, mid and high dose groups, respectively
- Prostate (males): 96% / 90%* / 105% versus ctrl in low, mid and high dose groups, respectively
- Seminal vesicle (males): 99% / 90%* / 102% versus ctrl in low, mid and high dose groups, respectively
*: p <= 0.05, **: p <= 0.01

The significant weight changes in cauda epididymides, kidneys and liver were regarded to be treatment-related, but not as adverse.

The weight decreases in prostate and seminal vesicles of the mid dose animals did not show a dose response-relationship and revealed no corresponding finding in histopathology of these organs in the high dose group animals. It was considered to be incidental and unrelated to treatment.

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

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation pups/litters
A few F1 pups showed spontaneous findings at gross necropsy, such discolored testis, dilated ureter, dilated pulmonary artery and dilated renal pelvis.
These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were considered not to be associated with the test substance.

Surplus F1 generation pups on PND 22
No abnormalities were detected during necropsy and gross pathology evaluation.

F1 rearing animals, Cohort 1A
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1A

The forestomach was the target organ in mid and high dose animals. There was a variety of findings ranging from erosions/ulcer through squamous cell hyperplasia (diffuse or focal) and hyperkeratosis (diffuse or focal). These findings were considered as treatment-related and adverse.

- Forestomach (females): erosions/ulcer in 1 low and 1 high dose animal
- Forestomach (males): squamous cell hyperplasia (diffuse / focal) in 0/5 mid and 5/11 high dose animals
- Forestomach (females): squamous cell hyperplasia (diffuse / focal) in 3/1 mid and 5/7 high dose animals
- Forestomach (males): hyperkeratosis (diffuse or focal) in 0/5 mid and 5/11 high dose animals
- Forestomach (females): hyperkeratosis (diffuse or focal) in 3/1 mid and 5/7 high dose animals

The erosion observed in a single low dose female animal was regarded to be incidental, as none of the other findings were observed, no dose-response relationship was present and regularly erosions in the rat stomach are observed also in control animals.

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.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation pups/litters

Thyroid hormones
No treatment-related alterations of T4 and TSH levels were observed in pups of all treatment groups at PND 4 and at PND22


F1 rearing animals, Cohort 1A

Estrous cycle data
-The mean estrous cycle duration was 4.0, 4.0, 4.2 and 4.1 days in control, low, mid and high dose group, respectively.
Estrous cycle data, revealed regular cycles in the females of all test groups and the mean estrous cycle duration was similar.

Thyroid hormones
No treatment-related alterations of T4 and TSH levels were observed in all treated animals at PND 90.
- T4 values were significantly increased in mid dose males.
Since the change was not dose dependent, it was regarded as incidental and not treatment related.

Spermanalysis
No treatment-related effects were observed 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.

Differential ovarian follicle count (DOFC)
No significant differences between the control and high dose group animals were observed

Absolute values
- Primordial: 6972 vs. 6827 in ctrl.
- Growing 638 vs. 686 in ctrl.
- Primordial + Growing 7610 vs. 7513 in ctrl.

Mean values
- Primordial: 348.60 vs. 341.35 in ctrl.
- Growing 31.9 vs. 34.3 in ctrl.
- Primordial + Growing 380.50 vs. 375.65 in ctrl.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no adverse TS related effects observed

Results: F2 generation

General toxicity (F2)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

F2 generation pups/litters

No test substance related adverse clinical signs were observed in any of the F2 generation pups of the different test groups.
One mid-dose male pup showed pale skin on PND 4.
One low-dose female pup showed a thread-like tail during the lactation and one low-dose pup showed kinked tail during PND 7 – 21. These observations were not considered to be associated with the test compound.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

F2 generation pups/litters
- Viability index indicating pup survival during early lactation (PND 0 - 4) varied between 98.7% / 99.5% / 99.1% and 98.8% in control, low, mid and high dose group, respectively.
- Lactation index indicating pup survival on PND 4 - 21 was 100% / 99.6% / 100% and 100% in control, low, mid and high dose group, respectively.

Thus, the test substance did not influence pup survival in any of the treated groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

F2 generation pups/litters
The mean body weights and body weight change of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

F2 generation pups/litters

Sex ratio
The sex distribution and sex ratios of live F2 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.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

F2 generation pups/litters
Anogenital distance/anogenital index
The anogenital distance and anogenital index of all test substance treated male and female pups were comparable to the concurrent control values.

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

F2 generation pups/litters
The number and percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13.
On PND 20 one single low-dose male pup exhibited two nipple/areola anlagen. This is considered to be a spontaneous event. No nipples/areolae were detected in any other male pup of all test groups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

F2 generation pups/litters

Pup organ weights
Absolute weights
The absolute pup organ weights (brain, thymus and spleen) of all test substance treated F2 pups were comparable to the concurrent control values.

Relative weights:
Following mean relative pup organ weight parameters were statistically significantly changed (*p≤0.05):
- Thymus (female): 105%, 109%* and 98% versus ctrl in low, mid and high dose groups, respectively
- Thymus (male+female): 105%, 107%* and 97% versus ctrl in low, mid and high dose groups, respectively

The increased relative thymus weights in the mid-dose group pups are regarded as an incidental finding, as the increase was marginal and not dose-related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F2 generation pups/litters

A few F2 pups showed spontaneous findings at gross necropsy, such discolored testis, small testis, discolored thymus, empty stomach, dilated renal pelvis, post-mortem autolysis, kinked tail, thread-like tail, hydroureter and hydronephrosis.
These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were considered not to be associated with the test substance.

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Effect levels (F2)

Dose descriptor:

NOAEL

Remarks:

Developmental

Generation:

F2

Effect level:

250 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no adverse TS related effects observed

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Results of the range-finding study and dose selection

The doses were selected based on signs of toxicity noted at dose levels of 100, 300, 500 and 1000 mg/kg bw/d in a previously conducted OECD 421 reproduction/developmental screening study (BASF project 80R0410/07R074) which preceded this definitive extended one generation reproductive toxicity study.

 Citral N was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d). As severe signs of intoxication were observed at 1000 mg/kg bw/d (particularly females, details see below) during the first days of administration (premating phase), the dose was lowered to 500 mg/kg bw/d from premating day 4 until the end of the study. Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (0.5% Sodium carboxymethyl cellulose (CMC) suspension in deionized water (with 5 mg/ 100mL Tween 80)).

 The duration of treatment covered about 4 weeks in-life period (males) including 2 weeks mating (mating pairs were from the same test group) as well as a 2 weeks premating period (females), 2 weeks mating period, the entire gestation, about 3 weeks of lactation period and about 5 weeks postmating period in females up to one day prior to the day of scheduled sacrifice of the animals.

 The 1000 mg/kg bw/d dose caused mortality (3 females) and severe clinical signs of intoxication (i.e. unsteady gait, severely reduced muscle tonicity and prone-position with abducted limbs) immediately after the start of treatment. After the high-dose was lowered to 500 mg/kg bw/d (premating day 4), still adverse clinical observations (hypothermia, unsteady gait, apathy, abdominal position, salivation, increased water consumption, decreased food consumption) were recorded, along with slight anemia, as well as a macroscopically visible thickening of the forestomach at necropsy. Histopathological examination of the forestomach in the 1000/500 mg/kg bw/d animals revealed a full thickness necrosis of the forestomach epithelium in the 3 female decedents, focal and transmural inflammation in 9 males and all females (most likely caused by erosion/ulceration that was not always visible on the slide), erosion/ulcer in 6 males and 6 females, as well as hyperplasia of the squamous epithelium and hyperkeratosis in 10 males and 7 females.

 The 300 mg/kg bw/d dose caused salivation and marginally decreased food consumption at the beginning of the study. Histopathological examination of the forestomach revealed inflammation in 8 males and 9 females, erosion/ulcer in 2 males and 2 females, hyperplasia of the squamous epithelium and hyperkeratosis in 10 males and 9 females, as well as focal degeneration of the squamous epithelium in1 female.

 In the 100 mg/kg bw/d dose group histopathological examination of the forestomach revealed inflammation in 1 male, hyperplasia of the squamous epithelium and hyperkeratosis in 1 male, as well as diffuse degeneration of the squamous epithelium in1 male.

 Thus, dose levels of 25, 80 and 250 mg/kg body weight/day were selected as dose levels for the present study. The high dose was intended to cause a similar adverse pathology as in the rangefinder while the low dose was intended to be a NOAEL. This procedure meets the principles of guidelines OECD 414 (adopted 2018) and OPPTS 870.3700 (US EPA), as well as ECHA practical guide 10 (“how to avoid unnecessary testing on animals”; chapter 4 “animal welfare”; ECHA-10-B-17-EN, 2010) which is in compliance with EU Directive2010/63/EU on animal protection.

 

Results of analyses

Stability analyses

The stability of test substance was demonstrated for a period of 7 days in the refrigerator.

Homogeneity analyses

The homogeneity of the mixtures was verified.

Concentration control analyses

Overall, measured values for Citral N were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the preparations. Some departures from the lower specification limit of 90 % were noted for single samples of Citral N in 0.5 % sodium carboxymethyl cellulose in drinking water with Tween 80 (0.5 mg/100 mL), which were found to be between 78.0 % - 89.4 % of their nominal concentrations. As these values were close to the lower specification limit and the vast majority of all measurements well met specification, those departures were considered not to have any influence on the validity of the present study.

Applicant's summary and conclusion

Executive summary:

Citral N was administered to groups of 25 male and 25 female healthy young Wistar rats asan aqueous preparation by stomach tube at different dosages (0, 25, 80 and 250 mg/kg body weight/day [mg/kg bw/d]). F0 animals were treated at least for 2 weeks prior to mating to produce a 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. Cohort 1B (=F1 generation parental animals) were selected to produce F2 pups. F1 animals selected for breeding were continued in the same dose group as their parents. Groups of 25 males and 25 females, selected from F1 pups to become F1 parental generation, were offered an aqueous preparation by stomach tube at different dosages (0, 25, 80 and 250 mg/kg bw/d) of the test substance post weaning, and the breeding program was repeated to produce a F2 litter. The study was terminated with the terminal sacrifice of the F2 weanlings and F1 parental animals. Control animals were dosed daily with the vehicle(0.5% Sodium carboxymethyl cellulose (CMC) suspension in drinking water (with 5 mg/ 100mL Tween 80)).

The parents' and the pups' state of health was checked each day, and parental animals were examined for their mating and reproductive performances. Water, food consumption and body weights of the F0 and F1 parents and F1 rearing animals was determined regularly and a detailed clinical observation (DCO) was performed in all F0 and F1 parents and F1 animals in cohorts 1A.

Estrous cycle data were evaluated for F0 and F1B parental females. In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A females for 2 weeks around PND 75. Moreover, the estrous stage of each female was determined on the day of scheduled sacrifice.

The F1 pups were sexed on the day of birth (PND 0) and were weighed on the first day after birth (PND 1) as well as on PND 4, 7, 14 and 21. Their viability was recorded. At necropsy, all pups were examined macroscopically. Date of sexual maturation, i.e. day of vaginal opening (females) or balanopreputial separation (males), of all F1 pups selected to become F1 parental and rearing animals was recorded.

All surviving F1 and F2 pups were examined for the presence or absence of nipple/areola anlagen on PND 13. If nipple/areola anlagen were recorded, all surviving male pups were carefully re-examined one PND 20. The number of nipple/areola anlagen were counted. Anogenital distance measurements were conducted on all live F1 and F2 male and female pups on PND 1.

Urine and blood samples for clinical pathological investigations were withdrawn from 10 selected F0 and cohort 1A animals per sex and group. Further blood samples were taken from a maximun of 10 surplus (culled) PND 4 pups per sex and group as well as from 10 surplus PND 22 pups per sex and group.

Various sperm parameters (motility, sperm head count, morphology) were assessed in the F0 generation males and cohort 1A males at scheduled sacrifice or after appropriate staining.

All F0 and F1 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to an extensive histopathological examination; special attention being paid to the organs of the reproductive system. A quantitative assessment of primordial and growing follicles in the ovaries was performed for all control and high-dose F1 rearing females cohort 1A. All F1 rearing animals were assessed by different pathological and histopathological examinations.

No test substance-related mortalities or adverse clinical observations were observed in any of the groups, indicating systemic toxicity. In particular, regularly conducted detailed clinical observations revealed no test substance-related adverse systemic effects.

Transient salivation during a short time period after gavage dosing was noted for nearly all high-dose and some mid-dose male and female animals (F0 and F1 animals across all cohorts) during all sections of the 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 not considered to be an adverse finding of systemic toxicity but may, however, had subsequent consequences, namely increases of food and water consumption. In the high-dose group (F0 and F1B animals) intermittent increases of food and water consumption were noted during all study segments. Similar changes, though to a much lesser extent, were also observed in the mid-dose group. Concurrent with food and water consumption the mean body weights and body weight gain of the high-dose group (F0 and F1B animals in particular) showed intermittent increases during several study segments. All these food/water consumption and body weight increases in the high-dose group were rather mild, in the lower dose groups they were even limited to short episodes in individual animals. Supposedly, the attempt of the animals to attenuate an unpleasant taste and/or smell after gavage dosing of the test material led to those phases of increased food/water consumption and their consequences. However, as an increase of food/water consumption was observed, the described changes were neither considered as adverse findings nor as signs of systemic toxicity.

Concerning clinical pathology, no treatment-related, adverse effects were observed in the parental F0 generation as well as in the offspring F1A rats up to a dose of the compound of 250 mg/kg bw/d.

Regarding pathology, the forestomach was the target organ in the F0 generation parental animals. The macroscopically observed thickening of the forestomach wall in males and thickening of the margo plicatus in females corresponded to hyperplasia (diffuse or focal) with hyperkeratosis (diffuse or focal) by light microscopy. Single animals revealed erosion/ulcer in addition which, in combination with the other findings, were seen as an aggravation of lesions. Mid and high dose male and female animals (80 and 250 mg/kg bw/d) were affected, with increasing number and severity in the high dose group (250 mg/kg bw/d).

The absolute liver weight in mid and high dose males (80 and 250 mg/kg bw/d) and kidney weight in high dose males (250 mg/kg bw/d) were above historical control data. These weight changes were assumed to be treatment-related but not adverse for the following reasons: no histopathologic correlate, no findings in clinical pathology, relative liver and kidney weights were within historical control data.

Male and female F1 generation rearing animals of cohort 1A revealed hyperplasia (diffuse or focal) with hyperkeratosis (diffuse or focal) in the forestomach. Single animals revealed erosion/ulcer in addition which, in combination with the other findings, were seen as an aggravation of lesions. Mid and high dose male and female animals (80 and 250 mg/kg bw/d) were affected with increasing number and severity in the high dose group (250 mg/kg bw/d).

High dose F1 males (250 mg/kg bw/d) revealed a significant increase of absolute and relative weight of cauda epididymis, kidney and liver; high dose F1 females (250 mg/kg bw/d) revealed a significant increase in absolute and relative weight of the liver. The altered weights were above historical control values. Nevertheless, these findings were regarded as treatment-related but not as adverse, because neither in histopathology nor in clinical pathology findings were observed which could explain the increased organ weights.

All other findings in the investigated internal organs of the F0 and F1A animals 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.

In the F1-generation cohort 1B males and females of the high dose group (250 mg/kg bw/d), the liver weights were significantly increased (males: absolute and relative, females: absolute only). In concordance with the discussion for the F0 and F1 cohort 1A animals these changes were regarded to be treatment-related but not as adverse.

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.

In the surplus F1-generation pups on PND 22 (F1 weanlings not selected for cohorts) neither treatment-related organ weight changes nor gross lesions were detected. Histopathology was not performed.

There were no indications from clinical examinations, that Citral N adversely affected the fertility or reproductive performance of the F0 and F1B parental animals up to and including the administered high-dose of 250 mg/kg bw/d. Estrous cycle data, sperm quality of males, mating behavior, conception, gestation, parturition, lactation and weaning were comparable between the rats of all groups including control and ranged within the historical control data of the test facility. The same is true for sexual organ weights and gross and histopathological findings of these organs in F0 and F1A females of all dose groups. Specifically, the results of the differential ovarian follicle count (DOFC) in F1A females – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – showed no significant differences between the control and the high dose group.

For all liveborn male and female pups of the F0 and F1B parents, no test substance-induced signs of developmental toxicity were noted at dose levels as high as 250 mg/kg bw/d. Postnatal survival, pup body weight gain as well as post-weaning development of the offspring until puberty remained unaffected by the test substance. Furthermore, clinical and/or gross necropsy examinations of the F1 and F2 pups revealed no adverse findings.

Measurement of thyroid hormones revealed no effect caused by the test substance, neither in the F0 parental animals nor in the liveborn F1 offspring.

Neither the anogenital distance/index nor the check for the presence of nipples/areolas, both very sensitive marker of potential endocrine-mediated imbalances, revealed any test substance-related effects.

Vaginal opening and preputial separation are commonly used developmental markers for onset of puberty in laboratory rats. No delays beyond a normal range of biological variation in rat (multi)generation studies which might be attributable to the treatment were noted in any of the test substance-treated groups.

Thus, under the conditions ofthe present extended one-generation reproduction toxicity studythe NOAEL (no observed adverse effect level) for general, systemic toxicity is 25 mg/kg bw/d, based on pathological findings in the gastrointestinal tract at the LOAEL of 80 mg/kg bw/d. The NOAEL for fertility and reproductive performance for the parental rats is250 mg/kg bw/d, the highest tested dose. The NOAEL for developmental toxicity in the F1 and F2 progeny is250 mg/kg bw/d, the highest tested dose.