Geraniol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to fertility:

- NOAEL: 800 mg/kg bw/day (OECD 443, oral)
- NOAEL: 300 mg/kg bw/day (OECD 421, dermal)

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity – with F2 generation and both developmental neuro- and immunotoxicity (Cohorts 1A, 1B with extension, 2A, 2B, and 3)

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)

GLP compliance:

yes (incl. QA statement)

Remarks:

Charles River Laboratories Edinburgh Ltd Elphinstone Research Centre Tranent, East Lothian EH33 2NE, United Kingdom

Limit test:

no

Justification for study design:

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

On November 19th, 2018 ECHA informed the REACH lead registrant that the submitted testing proposal was accepted (Decision number: TPE-D-2114449867-30-01/F). Pursuant, the registrant was requested to carry out:
"Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: OECD TG 443) in rats, oral route with the registered substance specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce systemic toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation."

On November 21st, 2018 and February 13th, 2019 the Biocidal Geraniol Task Force (BGTF) was informed by Anses (French agency for food, environment and occupational health safety Regulated Products Assessment Directorate) that the following study for the active substance Geraniol is required under the Biocidal Products Regulation (BPR, Regulation (EU) 528/2012):
"The extended one-generation reproductive toxicity study - EOGRTS – (OECD TG 443) with ten weeks premating exposure duration for parental (P0) animals could be performed, if an equivalent level of information than for a two-generation is provided.
We advise you to include all cohorts, especially since the data set on the developmental neurotoxicity and the immunotoxicity is not sufficiently robust in your dossier. That is to say:
- extension of Cohort 1B by mating of Cohort 1B animals to produce F2 generation (necessary for investigations on reproductive toxicity in offspring),
- Cohorts 2A and 2B (developmental neurotoxicity) and
- Cohort 3 (developmental immunotoxicity).
The rat is the preferred species, oral route of administration is the preferred route.
The highest dose level should be based on toxicity and selected with the aim to induce reproductive and/or other systemic toxicity."

Neither the REACH registrants nor the BGTF has been made aware by any official body that two different decisions on OECD 443 data requirements for the substance Geraniol were available at the same time. However, the registrants involved have made every effort to avoid double testing in the interest of animal welfare. The REACH lead registrant and BGTF members found a scientific way forward to ensure that the study design will be set-up in such a way that it covers both regulatory needs (REACH and Biocidal Products Regulation). Therefore, the requested OECD 443 was carried out as follows:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce systemic toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation;
- Cohorts 2A and 2B (developmental neurotoxicity) and
- Cohort 3 (developmental immunotoxicity).

The dose levels were selected based on a Reproduction/Developmental Toxicity Screening Study in Wistar Rats, Oral Administration (Gavage) with the registered substance Geraniol (BASF SE 2019; 80R0046/10R199) and the DECISION ON A TESTING PROPOSAL based on Article 40 of Regulation ((EC) No 1907/2006) for Geraniol (CAS 106-24-1).

Specific details on test material used for the study:

Expiration Date: 08 April 2022 (Re-test Date: 15 Mar 2022)
Density: 0.8810 g/mL
Purity: 99.2% (Dose calculations were not corrected for purity)
Storage Conditions: Ambient, protected from light

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Han Wistar rat was chosen as the animal model for this study as it is a rodent species accepted by regulatory agencies for reproductive toxicity testing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, UK
- Age at study initiation: F0 animals were approximately 6-7 weeks old (males) and approximately 5-6 weeks old (females)
- Weight at study initiation: F0 animals weighed between 160-261 g (males) and 106-157 g (females; target was minimum 125g but the lower starting weight was accepted at the time because the females were of the correct age and lower weight animals were present in all groups).
- Fasting period before study: no
- Housing: F0 animals were housed 2 or 3 per cage by treatment group and sex (unless reduced by mortality). A few days prior to mating, F0 males were transferred to individual cages. F0 females were transferred to these cages for mating. F0 females were transferred to individual solid bottomed cages after mating. F0 females with litters were retained in this type of cage until
termination. On a suitable day after completion of mating, the F0 males were re-housed with their original cage mates.
Cohort 1, 2 and 3 animals were housed 3 or 4 per cage by treatment group and sex (unless reduced by mortality). A few days prior to mating, Cohort 1B males were transferred to individual cages. F0 and Cohort 1B females were
transferred to these cages for mating.
Cohort 1B females were transferred to individual cages after mating. Cohort 1B females with litters were retained in this type of cage until termination. On a suitable day after completion of mating, the Cohort 1B males were re-housed with their original cage mates.
- Diet: SDS VRF-1 breeder diet (Special Diet Services, Essex, UK), ad libitum
- Water: water from the public supply, ad libitum
- Acclimation period: at least 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 26-66
- Air changes (per hr): >=10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations were prepared based on a method established at the Test Facility (Formulation Process Document 998849-19-110) at appropriate concentrations to meet dose level requirements. The test item was mixed with the vehicle using a magnetic stirrer and/or a high shear homogeniser until formulations were visibly homogeneous. The dosing formulations were prepared at least weekly and either delivered to the animal unit immediately, or stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and stirred for at least 30 minutes before, and then continuously during dosing.

Details on mating procedure:

F0 and Cohort 1B:
Pairing was on a 1 male to 1 female basis. Females were housed with their allocated co-group male partner during the evening (after 5 pm). For F0 animals, this commenced on Study Day 71 and for Cohort 1B animals this commenced between PND 100 and 116.
Vaginal lavages were taken early each morning from the day of pairing until mating had occurred and the stage of estrus observed in each vaginal lavage was recorded. The day of detection of a copulatory plug in situ and/or of sperm in the lavage was designated GD 0.
The pairing period for each pair of animals was a maximum of 14 nights.
If evidence of mating was not observed by the end of the pairing period, the female was separated from the male during the morning following the last night of pairing and treated as if mating had occurred during that night. Procedures for that female continued as if it had mated on the last night of pairing.
For each female the time taken to show a positive mating sign and the number of failed opportunities to mate (estruses passed without a sign of mating) were evaluated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis on Day 1, Week 14 and Week 25 (all test groups for concentration analysis, Test Group 2-4 for homogeneity).

Concentration Analysis
Duplicate top, middle and bottom (duplicate middle only for control) samples for each sampling time point were sent to the analytical laboratory. Triplicate top, middle and bottom (triplicate middle only for control) samples were retained at the Test Facility as backup samples. Sample volumes (0.1 mL) were collected into appropriately sized volumetric flasks and stored in a refrigerator set to maintain 4°C, protected from light. Concentration results were considered acceptable if sample concentration results were within or equal to ± 15% of
theoretical concentration. Each individual sample concentration result was considered acceptable if it was within or equal to ± 15%. For homogeneity, the criterion for acceptability was a relative standard deviation (RSD) of concentrations of <= 10% for each group. After acceptance of the analytical results, backup samples were discarded.

Stability Analysis
Stability analyses performed previously in conjunction with Lindsay, 2019, 423530 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for Lindsay, 2019, 423530.

Analyses were performed by ultra-performance liquid chromatography (UPLC) with ultra-violet detection using a validated analytical procedure (Lindsay, 2019, 423530).

Duration of treatment / exposure:

F0 Males: 10 weeks prior to mating and continuing throughout and after mating, until termination
F0 Females: 10 weeks prior to mating and continuing throughout mating and gestation, until at least Lactation Day (LD) 21
Cohort 1A: From PND 21 until termination
Cohort 1B Males: From PND 21, through mating and after mating until termination after the majority of females reached LD 21 (a small number of animals from each group were not dosed on PND 21).
Cohort 1B Females: From PND 21, through mating, pregnancy and littering until F2 pups reached PND 22-24
Cohort 2A: From PND 21 until termination
Cohort 3 (Groups 1 to 4): From PND 21-59

F2 pups were not dosed directly.

Frequency of treatment:

once daily

Details on study schedule:

F0 animals were randomly assigned to groups. Males and females were randomised separately.
The F0 animals were allowed to acclimate to the Test Facility rodent toxicology accommodation for a period of at least 2 weeks before the commencement of dosing.

Day 55, Animal 4516F – cannot be confirmed if animal was dosed

F0 and Cohort 1B females that were found to be in the process of littering, or had recently littered, at the time of scheduled dose administration, were not dosed on that day and dosing re-commenced the following day for these animals.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Test group 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Test group 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Test group 3

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Test group 4

No. of animals per sex per dose:

F0 Generation: 30/sex/group
F1 Generation:
Cohort 1A: One male and one female/litter (20/sex/group)
Cohort 1B: One male and one female/litter (25/sex/group)
Cohort 2A: One male or one female/litter (10/sex/group)
Cohort 2B: One male or one female/litter (10/sex/group)
Cohort 3: One male or one female/litter (10/sex/group)
F2 Generation: max. 25 litters per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
In an OECD 421 screening study in Wistar rats which used dose levels of 0, 100, 300 and 1000 mg/kg/day, there was no treatment-related mortality. However, body weight gain was reduced in males at 1000 mg/kg/day (by ~70%), with initial weight loss. Food consumption in females at 1000 mg/kg/day was reduced during the pre-mating period (by ~11%) and during lactation (~12%).
Clinical signs were reported in males at 100 mg/kg/day (noisy breathing [1/10] and ploughing [3/10]), at 300 mg/kg/day (ploughing [10/10]), and at 1000 mg/kg/day (noisy breathing [5/10], ploughing [10/10] and abdominal position [1/10]).
Clinical signs were reported during the pre-mating period in in females at 300 mg/kg/day (salivation [10/10]) and at 1000 mg/kg/day (salivation [10/10], unsteady gait [6/10], gasping [1/10], noisy breathing [6/10], piloerection [9/10], ploughing [9/10], reduced attention [6/10], apathy [1/10], abdominal position [5/10] and lateral position [1/10]). Clinical signs during gestation were seen in females at 100 mg/kg/day (salivation [9/10]), at 300 mg/kg/day (salivation [10/10] and ploughing [10/10]) and at 1000 mg/kg/day (salivation [9/9], ptosis [1/9], ploughing [9/9] and reduced attention [2/9]). Clinical signs during lactation were seen in females at 100 mg/kg/day (salivation [7/10] and ploughing [1/10]), at 300 mg/kg/day (salivation [8/8] and ploughing [7/8]) and at 1000 mg/kg/day (salivation [8/8] and ploughing [8/8]).
Decreased T4 values and increased serum TSH values in parental males.
Decreased T4 values in female PND13 pups.


- Fasting period before blood sampling for clinical biochemistry: yes

Positive control:

Developmental immunotoxicity examinations in cohort 3 animals.

For positive control animals (Cohort 3), cyclophosphamide was administered via oral gavage once daily on five consecutive days prior to necropsy (PND 51-55), at approximately the same time each day with a maximum of 6 hours difference between the earliest and the latest dose. Formulations were gently inverted prior to dosing. The dose volume for each animal was based on body weight measurement on the first day of treatment.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
F0 and F1 animals were observed twice daily, once at the start and once towards the end of the working day throughout the study for general health/mortality and moribundity.
F0 nimals were observed regularly throughout the day on each day of dosing (at least twice postdose) for signs of reaction to treatment, with particular attention being paid to the animals during the first hour after dosing. The onset, intensity and duration of any signs were recorded, as appropriate.
Cohort 1 and 2 animals were observed prior to dosing and when possible throughout the day of dosing for signs of reaction to treatment, with particular attention being paid to the animals during the first hour after dosing. Cohort 3 animals were also observed on the day of KLH administration, after injection. Cohort 3 positive control animals (Group 5) were observed during treatment with cyclophosphamide, at least once daily, up to the day prior to necropsy, for specific clinical signs after injection with cyclophosphamide. The time of onset, intensity and duration of any observed signs were recorded as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
F0 animals were subjected to detailed clinical observations at least weekly, beginning Week -1. Animals were removed from the cage for examination. The examinations included, but were not limited to, changes in skin, fur, eyes, mucous membranes, palpebral closure, vocalisation, rearing, arousal, stains and autonomic activity (lacrimation, salivation, piloerection, unusual respiratory pattern). Changes in gait and posture, as well as the presence of clonic or tonic movements, stereotypy or bizarre behaviour were assessed.
F1 Cohorts animals were subjected to detailed clinical observations at least weekly from weaning, starting on a suitable day within one week of weaning (PND 21) of all litters. Animals were removed from the cage for examination. The examinations included, but were not limited to, changes in skin, fur, eyes, mucous membranes, palpebral closure, vocalisation, rearing, arousal, stains and autonomic activity (lacrimation, salivation, piloerection, unusual respiratory pattern). Changes in gait and posture, as well as the presence of clonic or tonic
movements, stereotypy or bizarre behaviour were assessed.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 Males were weighed weekly beginning Week -1. Females were weighed weekly beginning Week -1 until pairing for mating and then at least on GD 0, 7, 14 and 20 and on LD 1, 4, 7, 14 and 21. Body weights taken on LD 0 were for dosing purposes only and have not been reported but retained in the study records. F1 pups were weighed individually on PND 1, 4, 7, 14 and 21. A weight was also recorded on the day of scheduled necropsy.
Cohorts 1A, 2A, 3 (both sexes) and Cohort 1B males were weighed weekly from weaning, starting on a suitable day within one week of weaning of the majority of litters (beginning of Nominal Week 4). Cohort 1B females were weighed weekly from this time until pairing for mating and then on GD 0, 7, 14 and 20 and on LD 1, 7, 14 and 21. Body weights taken on LD 0 were for dosing purposes only and have not been reported but retained in the study records. F2 pups were weighed individually, by sex, on PND 1, 4, 7, 14 and 21. Cohort 3 positive control animals (Group 5) were weighed once on the day of dosing with cyclophosphamide.

FOOD CONSUMPTION: Yes
Food consumption for F0 animals was quantitatively measured for both sexes weekly, beginning Week -1 until pairing for mating, for mated females on GD periods 0 to 7, 7 to 14 and 14 to 20 and on LD periods 1 to 7, 7 to 14 and 14 to 21. Food consumption was resumed weekly for males from Study Day 78 after mating and re-housing.
Food consumption was quantitatively measured weekly for Cohort 1A, 1B (both sexes, until pairing for mating), 2A and 3 animals, starting on a suitable day within one week of weaning of all litters (nominal Week 4), and for mated females (Cohort 1B only) on GD periods 0 to 7, 7 to 14 and 14 to 20 and on LD periods 1 to 7, 7 to 14 and 14 to 21. Food consumption was resumed weekly for Cohort 1B males on a suitable day after mating and re-housing.

WATER CONSUMPTION: Yes
Water consumption for F0 animals was monitored on a regular basis throughout the study by visual inspection of the water bottles. No intergroup differences were noted.

URINALYSIS: Yes
- F0 animals and Cohort 1A animals
- last week of dosing (10 animals/sex/group)
- Colour, Appearance/Clarity, Specific gravity, pH, Volume, Protein, Glucose, Bilirubin, Ketones, Blood

HAEMATOLOGY, COAGULATION AND CLINCICAL CHEMISTRY: Yes
- F0 animals and Cohort 1A animals
- morning of necropsy (10 animals/sex/group)
- Red blood cell count, Haemoglobin concentration, Haematocrit, Mean corpuscular volume, Red blood cell distribution width, Mean corpuscular haemoglobin concentration, Mean corpuscular haemoglobin, Reticulocyte count (absolute), Platelet count, White blood cell count, Neutrophil count (absolute), Lymphocyte count (absolute), Monocyte count (absolute), Eosinophil count (absolute), Basophil count (absolute), Large unstained cells (absolute)
- Activated partial thromboplastin time, Fibrinogen, Prothrombin time, Sample Quality
- Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Gamma-glutamyltransferase, Creatine Kinase, Total bilirubina, Urea, Creatinine, Calcium, Phosphate, Total protein, Albumin, Globulin, Albumin/globulin ratio, Glucose, Cholesterol, Triglycerides, Sodium, Potassium, Chloride, Sample quality, Total bile acids

BLOOD COLLECTION FOR TSH AND T4 MEASUREMENTS:
- F0 animals and Cohort 1A animals
- morning of necropsy (10 animals/sex/group)
- T4 was measured on Advia Centaur CP Immunoassay System by using solid phase, competitive chemiluminescent enzyme immunoassays. TSH was measured by using a sold phase enzyme immunometric assay – ELISA kit manufactured by BioVendor, Cat. No. RTC007R.

Oestrous cyclicity (parental animals):

Estrus Cycle Monitoring (F0 females):
Vaginal lavages were taken early each morning and the stages of estrus observed were recorded from 2 weeks prior to pairing until the day of detection of a copulatory plug in situ and/or of sperm in the lavage.
Vaginal smears were examined on the morning of necropsy to determine the stage of the estrus cycle to allow correlation with histopathology of reproductive organs, where this was considered necessary.

Estrus Cycle Monitoring (Cohort 1A and 1B):
Vaginal lavages were taken early each morning and the stages of estrus observed were recorded from the day after vaginal patency, continuing until the first confirmed estrus was determined (from approximately PND 75 for at least 14 consecutive days) for Cohort 1A females, and from 2 weeks prior to mating until detection of a copulatory plug in situ and/or of sperm in the lavage for Cohort 1B females. Vaginal smears were also examined on the morning of necropsy to determine the stage of the estrus cycle and to allow correlation with histopathology of reproductive organs, where considered necessary, for Cohort 1A females only.

Sperm parameters (parental animals):

F0 and Cohort 1A Males
- Computer Aided Sperm Assessment (CASA)
From all males, the right cauda epididymis was placed in 0.3% BSA in Medium 199 (as per Test Facility SOPs) and the sperm were allowed to “swim out” into the medium. An appropriate dilution of the sperm suspension was prepared and examined using a Hamilton Thorne sperm motility analyser.

- Sperm Count and Morphological Analysis
The cauda epididymis was minced and suspended. Dilutions of this sperm suspension were counted using a haemocytometer to obtain a total sperm count which was expressed per cauda epididymis and per gram of cauda epididymis.
From all samples of the sperm suspension described in the preceding paragraph, a sperm smear was prepared and stained with eosin Y solution. At least two hundred sperm per animal were evaluated for morphological abnormalities using criteria described by Wyrobek and Bruce (1975).

- Spermatid Count
The right testes were decapsulated and homogenised. The homogenate was sonicated to reduce tissue debris etc., if required. The number of homogenisation resistant spermatids in dilutions of this suspension were counted using a haemocytometer to obtain a total spermatid count which was expressed per testis and per gram of testis.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 10 pups/litter (5/sex/litter as nearly as possible) via random selection; excess pups were killed and discarded; when the total number of pups in a litter on PND 4 was ≤ 10 pups, no litter size adjustment occurred.

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD) on PND 1, pup weight on the day of AGD, presence of nipples/areolae in male pups on PND 13, gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS: YES
- Any pups that were found dead or were killed during lactation were sexed
and appropriately examined. Any externally abnormal decedent pup was preserved in 10% Neutral Buffered Formalin; externally normal pups were discarded.

WEANING AND SELECTION OF F1 ANIMALS FOR COHORTS 1A, 1B, 2A and 3:
From each group, 75 males and 75 females were selected at random on PND 21 and identified on that day for post-weaning assessments, nominally by selecting up to 5 males and 5 females from each litter. Where fewer than 60 litters were weaned, the necessary additional animals were obtained by selecting an additional pup from appropriate litters. These pups were removed from their mother on PND 21 and housed in their new cage.
Pups that were not selected for post-weaning assessments remained with their mother until termination.

ASSESSMENT OF SEXUAL MATURATION (COHORTS 1A, 1B, 2A and 3):
Commencing at PND 28, females were examined daily for vaginal opening. The day on which the vagina became open was recorded, as was the body weight on that day.
Commencing at PND 35, males were examined daily for balano-preputial separation.

BLOOD COLLECTION FOR TSH AND T4 MEASUREMENTS:
- F1 culled offspring (at least 2 pups/litter, where possible), PND 4 - at necropsy, T4 only
- F1 unselected pups (1 pup/sex/litter, where possible), at necropsy, TSH and T4
- T4 was measured on Advia Centaur CP Immunoassay System by using solid phase, competitive chemiluminescent enzyme immunoassays. TSH was measured by using a sold phase enzyme immunometric assay – ELISA kit manufactured by BioVendor, Cat. No. RTC007R.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY:
Assessment of Post-Natal Functional Development (Cohort 2A)
- Acoustic Startle: Between PND 23-25
- Motor Activity: Between PND 63-75
- Functional Observation Battery: Approximately PND 63-75
- Home Cage Observations: Posture/condition on first approach (animal undisturbed), checking for:, Prostration, Stereotypy / bizarre behaviour, Tremors (head, limbs, whole body), Convulsions, Ease of removal from the cage, Body temperature
- Handling Observations:
Pupillary function, Miosis / Mydriasis, Enophthalmos/ Exophthalmos, Lacrimation, Evaluation of diameter of the pupil, Body tone, Pinna response, Presence of salivation, Overall ease of handling, Respiration rate and pattern
- Air Righting
- Extensor Thrust
- Observations in a standardised arena (2 minute observation period): Rearing, Grooming, Urination and defecation, Arousal (level of alertness), Posture, Tremor (head, limbs, whole body), Convulsions, Piloerection, Palpebral closure, Gait abnormalities, Stereotypy (excessive repetition of behaviours) and/or unusual behaviours
- Functional Tests: Reaction to sudden sound (click above the head), Reaction to touch on the rump with a blunt probe, Grip strength, Pain perception, Landing Foot Splay, Other physical/functional abnormalities

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
KLH TDAR Assay – Cohort 3:
All animals were assessed for the ability to mount a T-cell dependent antibody response (TDAR) by measurement of circulating antibodies following administration of keyhole limpet haemocyanin (300 µg KLH on PND 51).
For Group 5, this immunisation was performed 2 to 4 hours after treatment of the positive control animals with cyclophosphamide on PND 51.
The animals were immunised by intravenous injection of KLH in 0.9% sodium chloride solution in the tail vein of each animal. Each immunisation was administered using sterile needles and disposable plastic syringes.
Blood samples (0.3 mL) were collected from the jugular vein using sterile hypodermic needles and plastic syringes before PND 51 and at PDN 55 or 56. Animals were not fasted prior to blood sampling.
Immediately after collection, the samples were transferred to glass tubes and allowed to stand at room temperature for at least 1 hour to allow complete coagulation. The samples were then centrifuged at approximately 1500 g at 4°C for 10 minutes and the serum separated into plain plastic tubes. The serum was then stored frozen at approximately -20°C until analysis for IgM antibodies to KLH was performed at the Test Facility. Samples were analysed by ELISA

Postmortem examinations (parental animals):

SACRIFICE
- Euthanised by exposure to carbon dioxide followed by exsanguination
- F0 Females: LD 22 or shortly thereafter
- F0 Males: After the majority of F0 females
- Cohort 2A and 2B animals underwent transcardial perfusion fixation

GROSS NECROPSY
Animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
The reproductive tracts of all F0 and Cohort 1B females were examined for signs of implantation, the number of any implantation sites being recorded. The total number of corpora lutea graviditatis was recorded for each female. The uteri of non-pregnant females were fixed in buffered formalin and stained using 10% aq (v/v) ammonium sulphide solution and examined for implantation sites.

For further information on ORGAN WEIGHTS, HISTOPATHOLOGY please refer to the attached document "Material and Methods - Information on Organ Weights, Tissue Collection and Preservation".

Immunophenotyping Sample Collection, Processing, and Analysis (Cohort 1A):
Spleen samples (approximately half of the spleen) were taken from 10 rats per sex per group of the Cohort 1A animals at necropsy and were collected into tubes containing RPMI-1640 medium and placed immediately on wet ice until they were processed. The remainder of the spleen was preserved in 10% neutral buffered
formalin.

HISTOLOGY
Identified tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with haematoxylin and eosin.

HISTOPATHOLOGY
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen.
Any cell- or stage-specificity of testicular findings was noted.
The kidneys of 5 control F0 generation males and 5 affected Group 4 F0 generation males
were stained with Mallory-Heidenhain stain.

OVARIAN FOLLICLE COUNTS (Cohort 1A)
From each ovary, 6 step serial sections at approximately 5 μm (a small amount into the formalin-fixed ovary e.g. 25 μm before the next section was taken) were taken. One section was stained with haematoxylin and eosin for routine evaluation and 5 sections stained for immunohistochemistry (IHC) staining using proliferating cell nuclear antigen (PCNA) marker for enumeration of primordial and primary follicles and for confirmation of the presence of or absence of the corpora lutea.

BONE MARROW SMEAR EVALUATION
Two bone marrow smears were taken from the femur of each euthanised F0 and Cohort 1A animal. Both bone marrow smears were air dried, fixed in methanol, stained with May-Grunwald-Giemsa stain and coverslipped. Bone marrow smears were not evaluated.

Postmortem examinations (offspring):

SACRIFICE
- Animals surviving until scheduled euthanasia were weighed, and euthanised by exposure to carbon dioxide followed by exsanguination.
- Animals less than 10 days old were killed by intraperitoneal injection of sodium pentobarbitone followed by exsanguination, by cervical dislocation. When possible, the animals were euthanised in a rotating order across dose groups such that similar numbers of animals from each group, including controls, were necropsied throughout the day.
- Culled PND 4 Pups from F1 and F2 Litters:
All culled pups at PND 4 of test group 1-4: Necropsy; Unscheduled Deaths: Necropsy
- Weanlings (unselected pups):
10 animals/sex/group, test group 1-4: Necropsy, Tissue Collection, Organ Weights

GROSS NECROPSY
Where practicable, offspring found dead or killed (unscheduled) were sexed internally, and then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any abnormal pups were, where practicable, fixed in 10% formalin or methylated ethyl alcohol, as appropriate. Externally normal pups were discarded.

For further information on ORGAN WEIGHTS, HISTOPATHOLOGY please refer to the attached document "Material and Methods - Information on Organ Weights, Tissue Collection and Preservation".

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and have been reported at the 1% and 5% levels.
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Any group with less than 3 observations was excluded from analysis.

Parametric/non-parametric statistical method:
- Levene’s test: The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test.
- Body Weight, Body Weight Gains, Food Consumption, Haematology Variables, Coagulation Variables, Clinical Chemistry Variables, Urinalysis Variables, Ovarian Scoring (total number of oocytes per animal), Organ Weights, Organ Weight Relative to Body Weight, Organ Weight Relative to Brain Weight, Sperm Morphology, Sperm Density and % Motility, Anogenital Distances (Litter Means), Motor Activity, FOB Quantitative Variables, Brain Morphometric Data, Litter Observations (Continuous) (e.g. Sex Ratio - Males, Mean Litter Body weights – total and sexes separate)

Non-parametric statistical method:
- Overall Kruskal-Wallis test: If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test.
- Pre-coital Interval, Day of Sexual Maturation, Natural Delivery and Litter Observations (Count) (e.g. Gestation Length, Live Pups, Implantation Sites), Developmental Landmark (Mean Day)

Incidence statistical method:
- Fisher’s exact test
- Pregnancy, Mating and Fertility Indices, Natural Delivery and Litter Observations
(Proportional) (e.g. Pregnant, Females with Liveborn, Gestation Index, Female with Liveborn)

Analysing Acoustic Startle:
SAS Software for Windows

Analysing TDAR data:
SAS v9.4, analysis was performed on the pooled (male and female) data

Reproductive indices:

Female Mating Index = Number of Females with Evidence of Mating (or no confirmed mating date and pregnant) / Number of Females Paired

Female Fertility Index = Number of Pregnant Females / Number of Females with Evidence of Mating (or no confirmed mating date and pregnant)

Female Pregnancy Index = Number of Pregnant Females / Number of Females Paired

Male Mating Index = Number of Males with Evidence of Mating (or female partner confirmed pregnant) / Number of Males Paired

Male Fertility Index = Number of Males Impregnating a Female / Number of Males with Evidence of Mating (or female partner confirmed pregnant)

Male Pregnancy Index = Number of Males Impregnating a Female / Number of Males Paired

Gestation Length = The gestation length was calculated from GD 0 to the day the first pup was observed.

Gestation Index = Percentage of pregnancies that result in birth of live litters:
Number of Animals with Live Offspring / Number of Animals Pregnant x 100

Offspring viability indices:

Birth Index (Percentage of pups born) = Total Number of Live and Dead Newborn Pups / Number of Implant Sites x 100

Live Birth Index (Percentage of pups born alive) = Number of Live Newborn Pups / Number of Live and Dead Newborn Pups x 100

Sex Ratio (% males) (Percentage of male pups per litter) = Number of Live Male Pup / Total Number of Live Pups x 100

Viability Index (Percentage of pups born that survive 4 days postpartum) = Number of Live Pups on Day 4 Postpartum / Number of Live Newborn Pups x 100

Lactation Index (Percentage of pups that survive 21 days postpartum) = Number of Live Pups on Day 21 Postpartum / Number of Live Pups on Day 4 (postculling) Postpartum x 100

Post-Implantation Loss/Litter = (Number of Implants – Total Newborn Pups) / Number of Implants x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A dose-related higher incidence and duration of transient salivation and ploughing behaviour in response to dosing was seen:
- in all males and females at 800 mg/kg/day, with a slightly lower incidence in females during gestation;
- in most females premating and males at 200 mg/kg/day and fewer females during gestation;
- in some females premating and males at 50 mg/kg/day – for a shorter duration - and no females during gestation. One or two control males showed these signs on a single day.

Red paws were noted in all males and females for several hours after dosing at 800 mg/kg/day on Day 8. Red ears were similarly noted for these animals and for all males and several females at 200 mg/kg/day between Days 8 and 10.

Treatment at 800 mg/kg/day was also associated with very low incidences/frequencies of (i) transient post dose abnormal (uncoordinated) gait, decreased activity/subdued behaviour, low carriage, erect fur and chewing action sporadically throughout the study; and (ii) increased respiration rate and hunched posture in females during the pre-mating period.

Other clinical observations were considered incidental due to their nature and/or distribution across the groups.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Two animals were euthanised for welfare reasons.
- F4509 (receiving 800 mg/kg/day) on Day 17: on removal from the cage for dosing, the animal showed increased respiration rate, decreased activity and was cold to touch with erected fur. It was not dosed and, half an hour later also had partly closed eyes, hunched posture and was subdued. Euthanasia was performed for welfare reasons.
At necropsy, cloudy red liquid was noted in the thoracic cavity; all other tissues were without
visible lesions. Histologically, a diffuse, mild infiltration by mixed cells in the lung was considered the cause of the animal’s terminal condition. It was possibly due to locally-deposited test item as a result of misdosing or reflux from previous days. The infiltrate included fibrinous exudate and macrophages with vacuolated cytoplasm. There was a mild mononuclear cell infiltrate in the pleura around the left lobe of the lung and adjacent to the sternum, and minimal inflammation in the thymic capsule. In the axillary lymph node there was a moderate accumulation of macrophages, some vacuolated, in the subcapsular sinus. This was possibly due to earlier misdosing. There were reactive changes in the mesenteric lymph node and vacuolation in the central nervous system, renal pelvis and splenic red pulp.
- F2527 (receiving 50 mg/kg/day) on Day 89: a toe injury sustained during gestation was successfully remedied under surgical anaesthesia. However, the animal was soon seen chewing her injured foot and with blood in the cage. She was therefore euthanised for welfare reasons. The only noteworthy finding was mild extramedullary haematopoiesis in the spleen.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related effects on adult body weight or weight gain during the
pre-mating period, gestation or lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: no test item-related effects, despite occasional statistically significant differences from the control group.

Females: At 800 mg/kg/day, statistically significant slightly higher food consumption values than
controls were noted intermittently during the pre-mating period (up to 15%) and for GD 14-20 (small at 9% but consistent with values up to 13% higher during GD 0-14). During lactation, intake was consistently slightly lower than controls (by 10 to 16%), attaining statistical significance for this group and for 200 mg/kg/day during Lactation Days (LD) 1-7 (-10%).

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Variation in haematology parameters, including some statistically significant differences between test item group and control group means, was considered normal. In all cases where statistical significance occurred (males: monocyte and reticulocyte counts), values were within the historical control range and not associated with changes in any other white or red blood cell parameters.

Coagulation: Despite some statistically significant differences from control in group mean values, these were generally small in magnitude and considered not to represent a test item effect.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

800 mg/kg/day: group mean values for the following parameters were higher in males and females than for the respective controls: bile acids, by 1.5- to 1.8-fold; ALP by approximately
1.4-fold and GGT by 1.5- to 1.7-fold. Intergroup differences for these parameters were not always statistically significant and the standard deviations for bile acids reflected wider ranges of values at 800 mg/kg/day than in the control group.

Variation in the remaining clinical chemistry parameters, including a statistically significant
difference from control for female globulin levels that was within the historical control range and not associated with any other protein changes, was considered normal. Creatinine kinase (CK) was 1.8-fold higher for females at this dose level but the standard deviation was large and there was no similar magnitude change in males.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Stimulating Hormone (TSH) levels were slightly higher than the concurrent control for F0 males and females at all dose levels (up to 1.5-fold at 50 mg/kg/day and approximately 2-fold at 200 and 800 mg/kg/day ). Group mean values were influenced by individual results that were below the reportable range and all listed as 0.63 ng/mL. Such values were likely due to a limit in assay sensitivity. However, the test item effect described above remained if these values were removed from the mean calculations. Individual values for all males and most females were within the historical control ranges, with only 1, 2 and 3 females at 50, 200 and 800 mg/kg/day respectively having values above these ranges compared with none for control group females.

There was no effect on circulating TSH values at PND 21. It was noted that all unselected (circa PND 21) female TSH values were below the reportable range, due to the limit in assay sensitivity.

Values for T4 were generally variable and did not suggest any effect of test item administration.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Despite some statistically significant differences from control in group mean values, these were considered to be small in magnitude and not representative of a test item effect.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were test item-related microscopic findings at 800 mg/kg/day in the nasal cavity in males (primarily degeneration in the olfactory epithelium and nasopharynx), thyroid in females (diffuse follicular cell hypertrophy), kidney in males (hyaline droplet accumulation) and spleen in males (increased haematopoiesis).
For further details please refer to "Any other information on results incl. tables".

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Females were cycling normally and pre-coital intervals were similar in all groups, with one female at 800 mg/kg/day having a pre-coital interval of 11 days – similar to that seen in an F1B control group female.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no clear dose-related trends in spermatid counts and differences from control were small, despite values attaining statistical significance at 50 and 800 mg/kg/day. It was therefore considered that there was no test item-related effect on these parameters and no effects on sperm counts, motility or morphology.

Reproductive performance:

no effects observed

Description (incidence and severity):

MATING FERTILITY, PREGNANCY INDICE:
similar in all groups.

GESTATION, LITTER PERFORMANCE AND SURVIVAL
- Gestation: no test item-related effects on the duration or the Gestation Index.
- Birth Index: The mean percentage of male pups per litter on LD 1 was almost identical in all groups.
- Live Birth Index: marginally lower at 800 mg/kg/day than in the control group (94.1% compared to 99.6%), with 3 litters at the highest dose level losing more than 1 pup – including two total litter losses by LD 1 –compared with none in the other groups. However, the values and incidences obtained in this high dose level group are not uncommon for control groups and were considered not to be an effect of the test item.
Pup survival between LD 0 and 4 was slightly lower at 800 mg/kg/day than in the control group, as shown by the Viability Index (85.9%, with several litters losing 2 or 3 pups, compared to 99.7% in the control group and no less than 97% for either generation in historical control data. Pup mortality occurred in 11 litters at 800 mg/kg/day compared with single litters at each of 0 (Control), 50 and 200 mg/kg/day.
Pup survival after the Day 4 cull was similar in all groups.
Pup and litter observations were also similar in all groups, with multiple nests and ‘scattering’ of pups noted for control and test item group litters.

Dose descriptor:

NOAEL

Remarks:

general, systemic toxicity

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

other: olfactory epithelium degeneration

Dose descriptor:

NOAEL

Remarks:

fertility/reproductive performance

Effect level:

800 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects observed at highest dose tested

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Note: the high dose level of 800 mg/kg/day was reduced to 600, then 400 mg/kg/day due to intolerance – see Mortality section - and titrated back via 600 to 800 mg/kg/day within the first two weeks of dosing. This dose level is simply referred to as 800 mg/kg/day.

A dose-related increase in incidence and duration of salivation and ploughing in response to dosing was seen in all cohorts: in most males and females at 800 mg/kg/day and in some males and females at 200 mg/kg/day. A small number of Cohort 2A control females and one Cohort 1B female at 50 mg/kg/day had salivation on isolated occasions.
Treatment at 800 mg/kg/day was associated with very low incidences/frequencies of transient post dose abnormal (uncoordinated) gait, decreased activity, low carriage, erected fur, chewing action, partially closed eyes, irregular respiration/breathing and pallor.
A small number of Cohort 1B animals at all dose levels were noted to have thinning fur/fur loss later on in the treatment period, compared with single control animals.
Other clinical observations were considered incidental due to their nature and/or distribution across the groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

In Cohort 1B, F4641 was euthanised on Day 1 of dosing (Post Natal Day (PND) 21), M4131 was euthanised on Day 2 (PND 22) and F2635 was found dead on Day 18 (PND 38). Both F4641 and M4131 were euthanised because they did not tolerate the high dose level. For M4131, Respiratory Rate Abnormal, Decreased; Fur, Erected; Eyes Closed, Partly; Low Carriage, and Activity Decreased were recorded 1-2 hours after dosing at 800 mg/kg/day. When the animal was also noted as being subdued, the decision was made to euthanise. For F4641, clinical signs were similar to those of other animals dosed at 800 mg/kg/day initially but worsened by 1 hour postdose and the animal was effectively moribund by approximately 2 hours post dose. The observations were Respiratory Rate Abnormal, Decreased/Irregular; Skin, Discoloured, Generalized, Pale; Activity Decreased/Abnormal Gait; Hunched Posture; Fur, Erected; Eyes Closed, Partly/Completely; Lying on Side/Prostrate, and Muscle Tone, Generalized, Decreased.
For F2635 receiving 50 mg/kg/day, this animal was found dead only 1 minute after dosing. A gavage dosing incident was suspected although there was no physical evidence of relevant trauma found at necropsy. In fact, none of these animals had any abnormalities at necropsy and they were not evaluated histologically.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

All Cohorts receiving 800 mg/kg/day (with the exception of Cohort 2B that was last weighed on PND 21) had slightly lower mean body weight values than concurrent control groups during Weeks 1 and 2 of dosing, which corresponded with the time period during which intolerance and mortality occurred. Differences from control were up to 18% for males and 13% for females, sometimes attaining statistical significance. Week 2 body weight for Cohort 3 males at 200 mg/kg/day was 11% lower than the concurrent control but, in isolation, this was considered not to be an effect of the test item.
Maternal body weight values during gestation and lactation (Cohort 1B) were essentially similar in all groups, even though this included statistically significant 6% higher weights on Gestation Day (GD) 20 at 200 and 800 mg/kg/day. Maternal weight gain from LD 1-21 was lower than control in a dose-related manner: -12 and -24% at 200 and 800 mg/kg/day respectively.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Consistently slightly higher food consumption values than controls were recorded for Cohort 1B males at 800 mg/kg/day from Week 16 (10-18%, statistically significant).
Gradually increasing slightly higher intake than controls was noted at 800 mg/kg/day for Cohort 1A females (9-15% between Weeks 7 and 10, statistically significant in the last week) and 1B females (10-20% between Weeks 6 and 13, statistically significant from Week 7).
These differences were not seen in Cohorts 2A and 3.
Slightly higher food consumption than controls was also seen throughout gestation (Cohort 1B): up to 16% at 200 and 800 mg/kg/day (statistically significant). Intake during lactation was similar in all groups.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Some white blood cell counts were higher in Cohort F1A test item groups than in controls, sometimes attaining statistical significance, in particular:
Basophils: 1.6-fold for females at 200 mg/kg/day and 1.7- to 2.1-fold for males and females at 800 mg/kg/day;
Monocytes: 1.6-fold for females at 800 mg/kg/day;
Large unstained cells: 1.6- to 1.7-fold for males and females at 800 mg/kg/day.
Variation in the remaining haematology parameters, including some statistically significant differences between test item group and control group means, was considered normal. In cases where statistical significance occurred (females: lymphocyte count, haematocrit), differences from control were small, values were within the historical control range and there were no associated red blood cell parameter changes.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid Stimulating Hormone (TSH) levels were slightly higher than the concurrent control for Cohort F1A males at all dose levels. The summary table lists these differences as approximately 2- to 3-fold. However, group mean values were influenced by individual results that were below the reportable range and all listed as 0.63 ng/mL. The test item effect described above remained if these values were removed from the mean calculations, giving differences of 1.5- to 1.7-fold. Most of the values below the reportable range were in the control group. Most of the individual measurable values in the test item groups and both of those in the control group were within the historical control ranges (combining F0 and F1 historical control data), with only 1 and 3 males at 200 and 800 mg/kg/day respectively having values above these ranges. It was noted that including F0 generation and similar age range historical control data resulted in comparing with animals older than those in the F1 generation, with a potential for inherently slightly higher TSH values in the historical control data.
There were few TSH values above the reportable range for females and a trend for higher values was therefore not suspected.
Values for T4 were generally variable and did not suggest any effect of test item administration.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Cohort F1A females in all groups reached their first estrus within similar day ranges. Most Cohort F1B females were cycling normally, except for F3630 at 200 mg/kg/day which appeared to enter a pseudopregnancy and one control group female (F1637) which did not cycle. These were considered typical anomalies to see within a set of females. Pre-coital intervals were similar in all groups, with one control female having a pre-coital interval of 13 days.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

In Cohort 1A at 800 mg/kg/day, total and per g testis spermatid counts were statistically
significantly 13-24% lower than the control value, also being just below the historical control data range. However, sperm counts were superior to control in the test item groups. The percentage of abnormal sperm was also higher at 800 mg/kg/day – the values still being very low, however: 0.43 and 1.18% compared with 0.30 and 0.78% in the control group). However, even this control group mean for abnormal sperm excluding tail defects was above the historical control data range (up to 0.2%). Motility was unaffected.

Reproductive performance:

no effects observed

Description (incidence and severity):

FERTILITY, PREGNANCY INDICES:
Male and female fertility and pregnancy indices were marginally lower at 200 and 800 mg/kg/day (between 83 and 88%) than the control group (96%). Considering F0 and F1 generation historical control data together – because the number of studies was limited - fertility indices were within this range; there being no historical control data for pregnancy indices. With the additional observation that the concurrent control group fertility indices were high compared to the historical control data, an effect of the test item was therefore not suspected.

DURATION OF GESTATION, LITTER PERFORMANCE AND SURVIVAL
There were no test item-related effects on the duration of gestation, Gestation Index or pup survival indices during lactation.
The mean percentage of male pups per litter on LD 1 was also unaffected, with no trends evident.
Pup and litter observations were generally similar in all groups, with multiple nests noted for control and test item group litters. While ‘scattering’ of pups was noted for some litters at all dose levels and not in the control group, it was recorded for control litters in the F0 generation. In the absence of other effects on F1 litters, this was considered to be an unfortunate distribution and not an effect of the test item.

OVARIAN FOLLICLE COUNTS
In Cohort 1A, total ovarian follicle counts at 800 mg/kg/day were lower than the control group, reflecting slightly lower primordial and primary follicle counts. However, these data are known to be quite variable, as can be seen from the historical control data range and a test item effect was therefore not suspected in this case. Corpora lutea.
were present in all females.

Dose descriptor:

NOAEL

Remarks:

general, systemic toxicity

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

other: olfactory epithelium degeneration

Dose descriptor:

NOAEL

Remarks:

fertility/reproductive toxicity

Effect level:

800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects observed at highest dose tested

Clinical signs:

effects observed, treatment-related

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In Cohort 1B, F4641 was euthanised on Day 1 of dosing (Post Natal Day (PND) 21), M4131 was euthanised on Day 2 (PND 22) and F2635 was found dead on Day 18 (PND 38).
Both F4641 and M4131 were euthanised because they did not tolerate the high dose level. For M4131, Respiratory Rate Abnormal, Decreased; Fur, Erected; Eyes Closed, Partly; Low Carriage, and Activity Decreased were recorded 1-2 hours after dosing at 800 mg/kg/day. When the animal was also noted as being subdued, the decision was made to euthanise. For F4641, clinical signs were similar to those of other animals dosed at 800 mg/kg/day initially but worsened by 1 hour postdose and the animal was effectively moribund by approximately 2 hours post dose. The observations were Respiratory Rate Abnormal, Decreased/Irregular; Skin, Discoloured, Generalized, Pale; Activity Decreased/Abnormal Gait; Hunched Posture; Fur, Erected; Eyes Closed, Partly/Completely; Lying on Side/Prostrate, and Muscle Tone, Generalized, Decreased.

For F2635 receiving 50 mg/kg/day, this animal was found dead only 1 minute after dosing. A
gavage dosing incident was suspected although there was no physical evidence of relevant trauma found at necropsy. In fact, none of these animals had any abnormalities at necropsy and they were not evaluated histologically.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean values for litter and pup weights were not significantly different, test item group values
being within 10% of control group values. The smallest litter and pup weights were recorded
at 800 mg/kg/day but the magnitude of difference from control was considered insufficient to
support a test item-related effect.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Consistently slightly higher food consumption values than controls were recorded for Cohort 1B males at 800 mg/kg/day from Week 16 (10-18%, statistically significant).
Gradually increasing slightly higher intake than controls was noted at 800 mg/kg/day for Cohort 1A females (9-15% between Weeks 7 and 10, statistically significant in the last week) and 1B females (10-20% between Weeks 6 and 13, statistically significant from Week 7).
These differences were not seen in Cohorts 2A and 3.
Slightly higher food consumption than controls was also seen throughout gestation (Cohort 1B): up to 16% at 200 and 800 mg/kg/day (statistically significant). Intake during lactation was similar in all groups.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Some white blood cell counts were higher in Cohort F1A test item groups than in controls,
sometimes attaining statistical significance, in particular:
- Basophils: 1.6-fold for females at 200 mg/kg/day and 1.7- to 2.1-fold for males and females
at 800 mg/kg/day;
- Monocytes: 1.6-fold for females at 800 mg/kg/day;
- Large unstained cells: 1.6- to 1.7-fold for males and females at 800 mg/kg/day.

Variation in the remaining haematology parameters, including some statistically significant differences between test item group and control group means, was considered normal. In cases where statistical significance occurred (females: lymphocyte count, haematocrit), differences from control were small, values were within the historical control range and there were no associated red blood cell parameter changes.

COAGULATION:
Despite some statistically significant differences from control in group mean values, these were generally small in magnitude and considered not to represent a test item effect.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Group mean values for bile acids were higher than controls by 1.9-fold for Cohort F1A males at 800 mg/kg/day and by 5.3-fold for Cohort F1A females at 200 and 800 mg/kg/day.
Differences from control attained statistical significance in all cases.
At 800 mg/kg/day, group mean values for ALT and ALP were also higher in males and females than for the controls: by 1.4- to 1.5-fold.
For females only, in comparison with control means, triglycerides were approximately 1.5-fold higher at 800 mg/kg/day and cholesterol was 1.3- to 1.4-fold higher at 200 and 800 mg/kg/day.

Variation in the remaining clinical chemistry parameters, including some statistically significant differences between test item and control group means, was considered normal. At 800 mg/kg/day, slightly but statistically significantly lower Na concentrations: by 0.99-fold, were on the limit or just outside the historical control range (see Appendix 54). However, the minor magnitude of this difference and the absence of any other test item-related changes in
electrolytes did not support this as a test item-related result. In other cases where statistical
significance occurred (males: urea and K concentrations; females: cholesterol concentration),
values were within the historical control range and there were no other associated clinical chemistry parameter changes. The significant result for female Na concentration at 50 mg/kg/day was not part of a dose-related trend and was within the historical control range.
Creatinine kinase was 1.3- to 1.9-fold higher for males at all dose levels but there was no dose-relationship and no similar magnitude change in females.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Despite some statistically significant differences from control in group mean values, these were considered to be small in magnitude and not representative of a test item effect.

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

Vaginal opening and balano-preputial separation were considered to be unaffected by the test item.
The age for preputial separation at 800 mg/kg/day was statistically significantly higher than the control group, at 42.2 days; however all groups were slightly above the historical control range (see Appendix 54) and the difference between all was less than 1 day.
Body weights at sexual maturation were essentially similar in all groups for males and for females separately. Despite a statistically significant difference in mean weight for females at 800 mg/kg/day compared with control, that was also below the historical control range at 99 g, the difference was small and the age for vaginal opening was within the historical control range.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distances were similar in all groups. Nipples were not retained in males

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The only general organ weight differences were noted in Cohort 1A animals at 800 mg/kg/day, where relative thyroid weight was higher than controls in females (all absolute thyroid weights were within the historical control range), absolute and relative kidney weight was higher than controls in males (2 of the 20 males at this dose level had absolute kidney weights greater than the historical control range), and absolute and relative liver weight was higher than controls in both sexes (1 of the 20 males and 8 of the 20 females at this dose level had absolute liver weights greater than the historical control range).
For further details please refer to "Any other information on results incl. tables".

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross pathology findings in any cohort.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

In Cohort 1A animals, in the nasal cavity, 1 female at 800 mg/kg/day had degeneration/regeneration of the nasopharyngeal epithelium. In the thyroid, at all dose levels
there was minimal, diffuse follicular cell hypertrophy (affecting both sexes). In the kidney of males at 800 mg/kg/day, and in 1 male at 200 mg/kg/day, there was hyaline droplet accumulation. In the liver at 800 and 200 mg/kg/day there was centrilobular hepatocyte
hypertrophy (affecting both sexes). In the spleen of males at 800 and 200 mg/kg/day there was minimal increased haematopoiesis.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

ACOUSTIC STARTLE: effects not treatment-related
Within Cohort 2A, for males, there were no statistically significant intergroup differences and, although average and maximum response amplitudes were lowest at 800 mg/kg/day, at approximately 13-14% below control values, this is quite a variable parameter by nature and the differences observed were slight. For females, statistical significance was not attained for these parameters and differences were generally less than 10% and not consistent across the 5 trials (sometimes lower, sometimes higher than control). Overall, it was considered that there was no effect of the test item on the acoustic startle response.
Latency to maximum response was not affected by the test item for males or females. Despite
a statistically significant difference in mean latency time for females at 800 mg/kg/day compared with control, the difference was small (4%) and all group means were below the historical control range.

MOTOR ACTIVITY: effects treatment-related
Within Cohort 2A, there were some lower levels of motor activity assessed at PND 63-75 for males at 800 mg/kg/day and for females at all dose levels, in a dose-related manner. The effect occurred in both sexes at 800 mg/kg/day, being more marked and attaining statistical significance for females. Statistical significance was also recorded for females at 200 mg/kg/day but not at 50 mg/kg/day where the magnitude of the effect was similar to that seen for high dose level males. These overall values also reflected changes seen in increasing numbers of individual sessions, from at least half at 50 mg/kg/day to most/all at 200 and 800 mg/kg/day.
For further details please refer to "Any other information on results incl. tables".

FOB: effects not treatment-related
In the Cohort 2A FOB assessed at PND 63-75, the low incidences of salivation and uncoordinated landing on air righting at 800 mg/kg/day were considered to be related to the clinical observations at this dose level. One male at this dose level had no pupil response when tested but, in isolation, this was considered of unlikely relationship to the test item.
There were no other notable qualitative results.
A small difference from control in mean forelimb grip strength was noted for males: -17% at 50 and 200 mg/kg/day, -14% accompanied by -11% for hindlimb grip strength at 800 mg/kg/day. Statistical significance was not attained. No other notable differences in quantitative parameters were seen.

NEUROPATHOLOGY: effects not treatment-related
At 800 mg/kg/day, Cohort 2A males and Cohort 2B and unselected pups of both sexes had lower brain weights; this was considered to be due to the lower body weight of the animals. There were no test item-related microscopic findings in Cohort 2A animals and no microscopic abnormalities were noted in Cohort 2B animals.

BRAIN MORPHOMETRY 2A/2B: effects not treatment-related
In Cohort 2A males, the brain morphometry measurements were less than controls at 800 mg/kg/day, by 2% to 8%. No effect was seen in females. A similar response was seen in the Cohort 2B males and females (2-9%). This was considered due to the lower body weight of the animals.
The mean length and width of the brain, as measured grossly, were similar to controls at all dose levels in Cohort 2A and were considered not to show a test item-relationship. In Cohort 2B males and females, the mean length and width of the brain at 200 and 800 mg/kg/day were lower than the control values by 6 to 16%. At 800 mg/kg/day, this was considered due to the lower body weight of the animals. Although at 200 mg/kg/day, body weight was similar to controls, brain weight at this dose level was only 3% less than controls in females, and was greater than controls in males. In view of the lack of effect on brain weight and the lack of statistical significance, the lower length and width of the brain at 200 mg/kg/day was considered not test item-related.

Developmental immunotoxicity:

no effects observed

Description (incidence and severity):

For all Cohort 1A splenic cell populations measured, there were no dose-dependent effects observed. Male animals in the control and 50 mg/kg/day dose groups had a higher percentage of B cells and lower percentage of T cells when compared with the 200 and 800 mg/kg/day dose groups. The 200 mg/kg/day and 800 mg/kg/day dose groups were comparable to all female dose groups, including the control, and showed no differences in percentage of B cells and T cells. Therefore, the difference noted in between the male groups could not be attributed to dosing with the test substance. The geomean of T helper cells percentage in male animals from the 800 mg/kg/day group was higher than that of the control group. However, this difference was marginal and the geomean was comparable to that of the female control group. There were no differences observed for the male Cytotoxic T cell or Natural Killer cell populations.

KLH TDAR Assay:
A clear humoral response to immunisation with KLH was observed in all animals; with KLH-specific IgM levels being greater on PND 56 than baselines values from before PND 51.
Mean levels of IgM were higher in female animals than in the male animals. There were no significant differences or dose-related changes in the concentration of KLH specific IgM antibodies in test-substance treated groups compared to the control groups. The test-susbtance-treated groups (Groups 2-4) and the 0 mg/kg/day negative control treated group (Group 1) showed a higher response than that of the 6 mg/kg/day Cyclophosphamide treated group, which was not unexpected as cyclophosphamide is a known immunosuppressant (significantly (p≤0.05) lower IgM levels at PND 56 in the positive control group compared to the negative control group).
The results indicated that the test-substance did not suppress the IgM response following administration when compared with the results obtained from the control animals.

Dose descriptor:

NOAEL

Remarks:

general, systemic toxicity (physical development until adulthood)

Generation:

F1

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: offspring viability, neonatal health, developmental status at birth

Dose descriptor:

NOAEL

Remarks:

developmental neurotoxicity

Generation:

F1

Effect level:

800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects observed at highest dose tested

Dose descriptor:

NOAEL

Remarks:

developmental immunotoxicity

Generation:

F1

Effect level:

800 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects observed at highest dose tested

Critical effects observed:

yes

Lowest effective dose / conc.:

800 mg/kg bw/day (actual dose received)

Organ:

other: olfactory epithelium

Treatment related:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Pup and litter observations were generally similar in all groups, with multiple nests noted for control and test item group litters. While ‘scattering’ of pups was noted for some litters at all dose levels and not in the control group, it was recorded for control litters in the F0 generation. In the absence of other effects on F1 litters, this was considered to be an unfortunate distribution and not an effect of the test item.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no test item-related effects on pup survival indices during lactation.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean values for litter and pup weights were not significantly different, test item group values
being within 10% of control group values. The smallest litter and pup weights were recorded
at 800 mg/kg/day but the magnitude of difference from control was considered insufficient to
support a test item-related effect.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

Anogenital distance data were similar in all groups. Nipples were not retained in males.

Other effects:

no effects observed

Description (incidence and severity):

There were no test item-related effects on the duration of gestation or Gestation Index.
The mean percentage of male pups per litter on LD 1 was also unaffected, with no trends evident.

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

800 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no effects observed at highest dose tested

Reproductive effects observed:

not specified

F0 generation: Organ weights

Table 1: Summary Group Mean Organ Weight Data – Scheduled Euthanasia (F0 males)

Group	1	2	3	4
Dose (mg/kg/day)	0	50	200	800
No. animals examined	30	30	30	30
Terminal body weight (g)	486.1	501.1	474.5	459.4
% difference from control	NA	+3.1	-2.4	-5.5
Thyroid Gland	(30)	(30)	(30)	(30)
Absolute value (g)	0.02148	0.02482	0.02312	0.02637^++
% difference from control	NA	+15.53142	+7.61831	+22.76183
% of body weight	0.00446	0.00494	0.00491	0.00576**
% difference from control	NA	+10.79192	+10.10130	+29.17267
% of brain weight	1.01944	1.15392	1.09583	1.25666^++
% difference from control	NA	+13.19164	+7.49390	+23.27061
Kidney	(30)	(30)	(30)	(30)
Absolute value (g)	2.6299	2.7613	2.7863	3.3248 ^++
% difference from control	NA	+4.9964	+5.9470	+26.4218
% of body weight	0.54436	0.55327	0.58890**	0.72606**
% difference from control	NA	+1.63632	+8.18195	+33.37799
% of brain weight	124.67748	128.26854	131.98862	158.48324**
% difference from control	NA	+2.88028	+5.86404	+27.11457
Liver	(30)	(30)	(30)	(30)
Absolute value (g)	14.6346	15.6058	15.3122	18.4433**
% difference from control	NA	+6.6359	+4.6299	+26.0250
% of body weight	3.01378	3.12248	3.23292**	4.01001**
% difference from control	NA	+3.60675	+7.27136	+33.05586
% of brain weight	693.16598	725.00322	725.57913	878.84113**
% difference from control	NA	+4.59302	+4.67610	+26.78654

Kruskal-Wallis & Dunn: ⁺ = p ≤ 0.05, ⁺⁺ = p ≤ 0.01 Anova & Dunnett: * = p ≤ 0.05, ** = p ≤ 0.01 NA: not applicable

 

Table 2: Summary Group Mean Organ Weight Data – Scheduled Euthanasia (F0 females)

Group	1	2	3	4
Dose (mg/kg/day)	0	50	200	800
No. animals examined	30	30	30	30
Terminal body weight (g)	258.0	263.9	263.6	262.7
Liver	(30)	(29)	(30)	(29)
Absolute value (g)	13.6516	13.8466	14.4242	15.8284*
% difference from control	NA	+1.4288	+5.6597	+15.9455
% of body weight	5.27498	5.19145	5.45229	6.00124^+
% difference from control	NA	-1.58338	+3.36140	+13.76810
% of brain weight	720.94328	712.09368	753.89009	829.49782*
% difference from control	NA	-1.22750	+4.56996	+15.5729

Kruskal-Wallis & Dunn: ⁺ = p ≤ 0.05, ⁺⁺ = p ≤ 0.01 Anova & Dunnett: * = p ≤ 0.05, ** = p ≤ 0.01 NA: not applicable

 

The only as adverse considered histopathological effect was the degeneration of the olfactory epithelium. The referring table is listed below.

Table 3: Microscopic Pathology (F0 Animals)

Removal Reason(s): TERMINAL EUTHANASIA Summary: Incidence	Male				Female
	0	50	200	800	0	50	200	800
	mg/kg/ day	mg/kg/ day	mg/kg/ day	mg/kg/ day	mg/kg/ day	mg/kg/ day	mg/kg/ day	mg/kg/ day
	Group1	Group2	Group3	Group4	Group1	Group2	Group3	Group4
Number of Animals	30	30	30	30	30	29	30	29
BODY CAVITY, NASAL Degeneration; olfactory epithelium ...mild ...moderate	0 0 0	0 0 0	0 0 0	2 1 1	0 0 0	0 0 0	0 0 0	0 0 0

 

F1 generation: Organ weights

Table 4: Summary Group Mean Organ Weight Data – Scheduled Euthanasia (F1 Cohort 1A males)

Group	1	2	3	4
Dose (mg/kg/day)	0	50	200	800
No. animals examined	20	20	20	20
Terminal body weight (g)	355.1	368.9	366.5	345.2
% difference from control	NA	+3.9	+3.2	-2.8
Kidney (No. weighed)	(20)	(20)	(20)	(20)
Absolute value (g)	2.3487	2.5471	2.5450	2.6141*
% difference from control	NA	+8.4474	+8.3601	+11.3022
% of body weight	0.66327	0.69344	0.69637	0.75965**
% difference from control	NA	+4.54796	+4.98892	+14.53094
% of brain weight	117.27415	125.03116	127.14442	133.07809**
% difference from control	NA	+6.61442	+8.41640	+13.47606
Liver (No. weighed)	(20)	(20)	(20)	(20)
Absolute value (g)	13.0374	13.7026	14.0452	15.7124**
% difference from control	NA	+5.1026	+7.7305	+20.5184
% of body weight	3.65644	3.69577	3.82116	4.53905**
% difference from control	NA	+1.07561	+4.50481	+24.13826
% of brain weight	651.22680	671.34847	702.84981	799.75331**
% difference from control	NA	+3.08981	+7.92704	+22.80719

Anova & Dunnett: * = p ≤ 0.05, ** = p ≤ 0.01 NA: not applicable

 

Table 5: Summary Group Mean Organ Weight Data – Scheduled Euthanasia (F1 Cohort 1A females)

Group	1	2	3	4
Dose (mg/kg/day)	0	50	200	800
No. animals examined	20	20	20	20
Terminal body weight (g)	212.2	212.0	216.0	209.6
% difference from control	NA	-0.1	+1.8	-1.2
Thyroid Gland	(20)	(20)	(20)	(20)
Absolute value (g)	0.01553	0.01677	0.01627	0.01800
% difference from control	NA	+8.01017	+4.78983	+15.90000
% of body weight	0.00732	0.00793	0.00758	0.00870*
% difference from control	NA	+8.44613	+3.64023	+18.84588
% of brain weight	0.82952	0.89298	0.86836	0.98988**
% difference from control	NA	+7.65002	+4.68199	+19.33080
Liver (No. weighed)	(20)	(20)	(20)	(20)
Absolute value (g)	7.9172	8.0360	8.6843	10.4670**
% difference from control	NA	+1.5012	+9.6891	+32.2060
% of body weight	3.71811	3.78564	4.01365	4.98153**
% difference from control	NA	+1.81621	+7.94848	+33.98002
% of brain weight	423.60244	428.49490	463.96970	575.46120**
% difference from control	NA	+1.15496	+9.52952	+35.84936

Anova & Dunnett: * = p ≤ 0.05, ** = p ≤ 0.01 NA: not applicable

Other noteworthy organ weight differences are detailed in the Pathology phase report and were considered not test item-related.

 

F1 generation: Motor activity

Table 6: Motor activity

Dose level (mg/kg/day)	Female	Female	Male	Female
	50	200	800	800
Basic Movement	-	-14	-15	-24
Fine Movement	-	-	-16	-17
X Ambulation	-12	-26	-	-39
Y Ambulation	-19	-33	-16	-43

A dash (-) indicates absence of a test item-related change. Numerical values indicate the percentage difference of group mean values from the Overall control group.

Bold values were statistically significant from the control group (P ≤ 0.05/0.01).

Effect on fertility: via oral route

Dose descriptor:

NOAEL

800 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

Dose descriptor:

NOAEL

300 mg/kg bw/day

Species:

rat

Additional information

General information:

On November 19th, 2018 ECHA informed the REACH lead registrant that the submitted testing proposal was accepted (Decision number: TPE-D-2114449867-30-01/F). Pursuant, the registrant was requested to carry out:

"Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: OECD TG 443) in rats, oral route with the registered substance specified as follows:

- Ten weeks premating exposure duration for the parental (P0) generation;

- Dose level setting shall aim to induce systemic toxicity at the highest dose level;

- Cohort 1A (Reproductive toxicity);

- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation."

 

On November 21st, 2018 and February 13th, 2019 the Biocidal Geraniol Task Force (BGTF) was informed by Anses (French agency for food, environment and occupational health safety Regulated Products Assessment Directorate) that the following study for the active substance Geraniol is required under the Biocidal Products Regulation (BPR, Regulation (EU) 528/2012):

"The extended one-generation reproductive toxicity study - EOGRTS – (OECD TG 443) with ten weeks premating exposure duration for parental (P0) animals could be performed, if an equivalent  level of information than for a two-generation  is provided. 

We advise you to  include all cohorts, especially since the data set on the developmental neurotoxicity and the immunotoxicity is not sufficiently robust in your dossier. That is to say:

- extension of Cohort 1B by mating of Cohort 1B animals to produce F2 generation (necessary for investigations on reproductive toxicity in offspring),

- Cohorts 2A and 2B (developmental neurotoxicity) and

- Cohort 3 (developmental immunotoxicity).

The rat is the preferred species, oral route of administration is the preferred route.

The highest dose level should be based on toxicity and selected with the aim to induce reproductive and/or other systemic toxicity."

 

Neither the REACH registrants nor the BGTF has been made aware by any official body that two different decisions on OECD 443 data requirements for the substance Geraniol were available at the same time. However, the registrants involved have made every effort to avoid double testing in the interest of animal welfare. The REACH lead registrant and BGTF members found a scientific way forward to ensure that the study design will be set-up in such a way that it covers both regulatory needs (REACH and Biocidal Products Regulation). Therefore, the requested OECD 443 was carried out as follows:

- Ten weeks premating exposure duration for the parental (P0) generation;

- Dose level setting shall aim to induce systemic toxicity at the highest dose level;

- Cohort 1A (Reproductive toxicity);

- Cohort 1B (Reproductive toxicity) with extension to mate the Cohort 1B animals to produce the F2 generation;

- Cohorts 2A and 2B (developmental neurotoxicity) and

- Cohort 3 (developmental immunotoxicity).

 

Available data on Geraniol:

Reproductive toxicity was evaluated in a study performed according to OECD Guideline 443 (Charles River, 2021). The objective of this study was to determine the potential toxicity of the test chemical when given by oral gavage to adult rats and their offspring. Dosing of animals can be summarised as follows: F0 animals from 10 weeks prior to mating continuously until termination, F1 animals from Post Natal Day 21 (except Cohort 2B, which was not dosed) continuously until termination.

This study was designed to provide an evaluation of the pre- and postnatal effects on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and their offspring. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, physical and functional development until adulthood, and nervous and immune system development was undertaken to identify specific target organs in the offspring. In addition, the study provided information about the effects of the test substance on the integrity and performance of the adult male and female reproductive systems.

For the F1 generation, the highest dose level of 800 mg/kg/day was reduced to 600, then 400 mg/kg/day due to intolerance (mortality) and titrated back via 600 to 800 mg/kg/day within the first two weeks of dosing (by Post Natal Day 33). This dose level is simply referred to as 800 mg/kg/day.

The following parameters and end points were evaluated in this study: clinical observations, body weights, food consumption, estrus cycles, mating performance, fertility, duration of gestation, litter performance and survival, litter and pup weights, pre-weaning physical development, sexual maturation, post-natal functional development, clinical pathology parameters, thyroid hormones, KLH TDAR assay, immunophenotyping, gross necropsy, organ weights, brain morphometry, neuropathology, sperm evaluation, ovarian follicle counts and histopathological examinations.

Two F1 weanlings were euthanised on Day 1 or Day 2 of dosing due to clinical signs of intolerance to dosing at 800 or 600 mg/kg/day.

Compared to control animals:

Transient salivation and ploughing behaviour occurred in a dose-related manner in response to dosing in F0 and F1 animals at 800 and 200 mg/kg/day, and in F0 animals at 50 mg/kg/day.

Red paws (all) were noted for all F0 animals for several hours after dosing at 800 mg/kg/day on Day 8 only. Red ears were similarly noted for these animals and for most animals at 200 mg/kg/day between Days 8 and 10.

Treatment of F0 and F1 animals at 800 mg/kg/day was also associated with very low incidences/frequencies of transient post dose abnormal (uncoordinated) gait, decreased activity/subdued behaviour, low carriage, erected fur and chewing action. Some of these signs are consistent with the published observation that Geraniol is a CNS depressant (Medeiros et al 2018). Partially closed eyes, irregular respiration/breathing and pallor were also observed in F1 animals only. In addition, a small number of F1 animals at all test item dose levels had thinning fur/fur loss later on in the treatment period.

F1 animals at 800 mg/kg/day had slightly lower body weight values (up to 18% for males and 13% for females) during Weeks 1 and 2 of dosing, which corresponded with the time period during which intolerance and mortality occurred. Maternal weight gain from LD 1-21 was also lower: -12 and -24% at 200 and 800 mg/kg/day respectively.

At 800 mg/kg/day, slightly higher food consumption was noted for F0 females intermittently during the pre-mating period (up to 15%) and GD 14-20 (minimal at 9% but consistent with values up to 13% higher during GD 0-14). During lactation, intake was consistently slightly lower (by 10 to 16%), also for 200 mg/kg/day during LD 1-7 (by 10%).

At 800 mg/kg/day, slightly higher food consumption was recorded for Cohort 1B males from Week 16 and for Cohort 1A and 1B females between Weeks 6 and 13 (9-20%). Slightly higher food consumption was also noted throughout gestation (Cohort 1B): up to 16% at 200 and 800 mg/kg/day.

At 800 mg/kg/day, the following clinical chemistry parameters were slightly higher in F0 animals: bile acids, by 1.5- to 1.8-fold; ALP by approximately 1.4-fold and GGT by 1.5- to 1.7-fold.

The following clinical pathology parameters were higher in F1 animals: Basophils: 1.6-fold for females at 200 mg/kg/day and 1.7- to 2.1-fold at 800 mg/kg/day; Monocytes: 1.6-fold for females at 800 mg/kg/day; Large unstained cells: 1.6- to 1.7-fold at 800 mg/kg/day; bile acids, by 1.9-fold for males at 800 mg/kg/day and by 5.3-fold for females at 200 and 800 mg/kg/day; at 800 mg/kg/day, ALT and ALP by 1.4- to 1.5-fold; for females only, triglycerides approximately 1.5-fold higher at 800 mg/kg/day and cholesterol 1.3- to 1.4-fold higher at 200 and 800 mg/kg/day.

TSH was slightly higher for F0 animals and for Cohort 1A males at all dose levels (by approximately 1.5- to 2-fold). Despite numerous results being below the reportable range and most measurable values being within historical control data ranges, a small number of individual values above the historical control ranges were recorded particularly at 800 mg/kg/day.

F0 pup survival between LD 0 and 4 was lower at 800 mg/kg/day (Viability Index 85.9%, with several litters losing 2 or 3 pups), with pup mortality occurring in 11 litters compared with 1 litter in each of the other groups, including control.

Some lower levels of motor activity were recorded for F1 males at 800 mg/kg/day (-15 to -16%, not statistically significant) and for females at all dose levels, in a dose-related manner: -12 to -19% ambulation only at 50 mg/kg/day (not statistically significant), -14 to -33% and -17 to -43% at 200 and 800 mg/kg/day respectively. These values also reflected changes seen in increasing numbers of individual sessions, from at least half at 50 mg/kg/day to most/all at 200 and 800 mg/kg/day.

In the F1 generation Functional Observation Battery (FOB), low incidences of severe salivation, with none in the control group, and uncoordinated landing on air righting at 800 mg/kg/day were considered to be related to the clinical observations at this dose level. A small difference in mean forelimb grip strength was noted for males: -17% at 50 and 200 mg/kg/day, -14% accompanied by -11% for hindlimb grip strength at 800 mg/kg/day (no statistical significance).

For F0 animals, thyroid, liver and kidney weights were higher (between 23 and 33%) in males at 800 mg/kg/day, with liver weights in females also higher (14 to 16%) at this dose level. In Cohort 1A animals at 800 mg/kg/day, thyroid weight was higher (16 to 19%) in females, kidney weight was higher (11 to 15%) in males, and liver weight was higher (between 21 and 36%) in both sexes. At 800 mg/kg/day, Cohort 2A males, Cohort 2B and F1 and F2 unselected pups of both sexes had lower brain weights; this was considered to be due to the lower body weight of the animals. Cohort 2A male and Cohort 2B brain morphometry measurements were less (2 to 9%) and Cohort 2B brain length and width were lower (6 to 12%) for the same reason.

In the F0 generation, test item-related microscopic findings at 800 mg/kg/day occurred in the nasal cavity in males (primarily degeneration in the olfactory epithelium and nasopharynx), thyroid in females (diffuse follicular cell hypertrophy), kidney in males (hyaline droplet accumulation) and spleen in males (increased haematopoiesis).

In Cohort 1A, in the nasal cavity, 1 female at 800 mg/kg/day had degeneration/regeneration of the nasopharyngeal epithelium. In the thyroid, at all dose levels there was minimal, diffuse follicular cell hypertrophy (affecting both sexes). In the kidney of males at 800 mg/kg/day, and in 1 male at 200 mg/kg/day, there was hyaline droplet accumulation. In the liver at 800 and 200 mg/kg/day there was centrilobular hepatocyte hypertrophy (affecting both sexes). In the spleen of males at 800 and 200 mg/kg/day there was minimal increased haematopoiesis.

At 800 mg/kg/day, total and per g testis spermatid counts for Cohort 1A were 13-24% lower. The percentage of abnormal sperm was also higher at 800 mg/kg/day – the values still being very low, however: 0.43 and 1.18% compared with 0.30 and 0.78% in the control group.

There were no test item-related effects on the following parameters or endpoints:

F0 generation adult survival, body weight, haematology, coagulation, urinalysis, estrus cycles and mating performance, duration of gestation, litter performance, gross pathology, sperm evaluation.

F1 generation coagulation, urinalysis, estrus cycles and mating performance, duration of gestation, litter survival and performance, anogenital distance, nipple retention, vaginal opening, balano-preputial separation, acoustic startle, gross pathology, neuropathology, spleen immunophenotyping, ovarian follicle counts. The test item did not suppress the IgM response.

For further details on developmental toxicity please refer to the section "Developmental toxicity / teratogenicity".

In terms of the study objectives, it was concluded that administration of Geraniol by daily oral gavage administration was not initially tolerated by F1 weanling rats at dose levels of greater than 400 mg/kg/day, being associated with mortality at 600 and 800 mg/kg/day. Target organ effects were primarily adaptive in nature, affecting liver, thyroid and spleen at dose levels of 800 mg/kg/day (F0 generation) and from 50 mg/kg/day (F1 generation). However, the olfactory epithelium degeneration at 800 mg/kg/day was an adverse change because it involved a neural tissue that does not regenerate. Based on the results, the following no-observed-adverse-effect levels (NOAELs) were assigned:

Systemic toxicity for adult rats = 200 mg/kg/day (based on olfactory epithelium degeneration)

Fertility/reproductive performance = 800 mg/kg bw/day (no effects observed up to the highest dose tested)

 

According to Cadby et al. (Consumer Exposure to Fragrance Ingredients: Providing Estimates for Safety Evaluation, Regulatory Toxicology and Pharmacology 36, 246 - 252 (2002)) the dermal route is the most relevant of systemic exposure to fragrance materials. "Safety evaluation of fragrance ingredients is based on data from tests, in which material is administered to human or animal subjects by placing it on the surface of the skin. For this reason, it is unnecessary to consider anything further than estimating quantities of these ingredients deposited on the surface of the skin." For this reason and according to REACh-Annexes ("most appropriate route of administration, having regard to the likely route of human exposure") a dermal reproduction / developmental toxicity screening test according to OECD 421 has been performed in wistar rats (BASF SE, 2010).

A 10 day range finding study with 3 female rats per dose using doses of 1000, 750, 500 and 300 mg/kg bw/day led to different grades of skin irritation (scales and erythema) in the doses of 500 - 1000 mg/kg bw/d, and no clinical findings of the treated skin in the 300 mg/kg bw/day group. Based on these findings, doses of 0, 50, 150 and 450 mg/kg bw/day were chosen for the reproduction screening study. The test substance was administered to 10 male and 10 female young Wistar rats dissolved in corn oil. Application area was the intact clipped skin of the back (dorsal and dorsolateral areas of the trunk; not less than 10% of the body surface). The first clipping was carried out at least 24 hours before the randomization. The rats were reclipped at least once a week (depending on the hair growth). Dermal application of the test-substance preparations to the clipped intact dorsal skin was carried out with 3-mL syringes and a semiocclusive dressing (4 layers of absorbent gauze and stretch bandage). The test-substance preparation was applied to the dorsal skin with the syringe in each case. After removal of the dressing, the application area was washed with lukewarm water. Application was daily for at least six hours.

During the course of the study the initial high dose (450 mg/kg bw/day) turned out to be intolerable for the rat skin (strong irritation reactions), so that from day 10 onwards the high dose had to be reduced to 300 mg/kg bw/day. About 2 weeks after the beginning of treatment, animals were mated to produce a litter. Mating pairs were from the same dose group. Pregnant females were allowed to give birth and the offspring was brought up until postnatal day (PND) 4. The study was terminated with the sacrifice of the pups on PND 4 and of lactating dams shortly thereafter.

Regarding clinical examinations, only signs of local dermal toxicity were observed for males and females at all dose levels. No changes in food consumption and body weight data were seen at any dose level.

Fertility indices for male and female animals were not impaired by test-substance administration.

Regarding pathology, there were no treatment-related necropsies or histological findings in ovaries, testes or epididymides associated with dermal administration of the test substance. The local minimal inflammatory reactions in the skin of treated males (test groups 1-3) and females (test group 3 only) were regarded as related to treatment and adverse.

Therefore, the NOAEL for fertility for Geraniol is shown to be >300 mg/kg bw/day via the dermal route. Systemic exposure to geraniol is limited by the strong local irritation effects.

 

Additional studies:

Apart from studies with the test substance geraniol, studies using structurally similar compounds were taken into account to address the endpoint of toxicity to reproduction. Geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol) and nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) are trans/cis-isomers and therefore differ only in their relative orientation of functional groups within the molecule. Furthermore, relevant physicochemical parameters show comparability between geraniol and nerol.

A study according to OECD 421 using the oral route of exposure was performed with the reaction mass of geraniol and nerol (E- and Z-isomer, 60:40 -mixture) (BASF SE, 2010).

The test substance was administered to groups of 10 male and 10 female young Wistar rats dissolved in corn oil, via daily gavage. The dose levels were 0, 100, 300 and 1000 mg/kg body weight/day. About 2 weeks after the beginning of treatment, animals were mated to produce a litter. Mating pairs were from the same dose group. Pregnant females were allowed to give birth and the offspring was brought up until postnatal day (PND) 4. The study was terminated with the sacrifice of the pups on PND 4 and of lactating dams shortly thereafter.

All mid- and high-dose as well as some low-dose animals of both sex showed transient salivation for a few minutes immediately after each treatment. This was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is neither considered to be a sign of systemic toxicity nor as adverse.

Clinical observations indicated distinct toxicity in the exposed parental animals of the high dose group (1000 mg/kg bw/d) but not in the animals of the mid- and low-dose group.

A reduction of food consumption (up to 10% in males and females during treatment weeks 0-1 as well as females (-34%) during lactation), and decreased body weight in males (up to -5%) had been determined during treatment weeks 2-4. A similar pattern as for clinical observations was noted for body weight and body weight change of the parental animals. A distinct decrease was noted in the high-dose animals of both sex even manifested in different time periods of the study. The body weight change in males was reduced from week 0 to 5 (-28% on average in this time period). In females a significant body weight change was observed during lactation leading to a body weight loss (-3%). Consequently, the body weight was decreased in males during treatment weeks 2-5 (-7%) and in females of week 6 (-9%).

The test compound did not adversely affect fertility of the F0 generation parental animals at all dose levels as there were no changes of male/female mating and fertility indices, time until successful copulation, duration of pregnancy and mean number of implantations.

Since the reaction mass of geraniol and nerol consists of ca. 60% geraniol and 40% nerol, it was concluded that a systemic exposure to 600 mg/kg bw/day of geraniol can be regarded as NOAEL concerning effects on fertility.However, the NOAEL for systemic effects is lower.

 

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD TG 422) has been performed (DRT, 2013). The test item nerol was administered by dietary admixture (initially mixed with 2% corn oil) to three groups of Han Wistar rats for up to 42 consecutive days (main phase males, toxicity females and recovery animals) or between 41 and 53 days (including three weeks exposure phase, pairing, gestation and early lactation; main phase females) at dietary concentrations of 3000, 6000 and 12000 ppm (equivalent to 191.2, 374 and 720 mg/kg bw/day, respectively). A control group was treated with basal laboratory diet (with 2% corn oil). Two recovery groups, each of five males and five females, were treated with the high dose (12000 ppm) or basal laboratory diet alone for 42 consecutive days and then maintained without treatment for a further 14 days. Pairing of main phase animals and recovery males within each dose group was undertaken on a one male:one female basis within each treatment group on Day 22 of the study, with females subsequently being allowed to litter and rear their offspring to Day 7 of lactation.

No findings, which were considered to be test item-related, were noted during clinical observations, functional observational battery testing or locomotor activity testing in males or females at any dose level.Visual inspection of water bottles did not reveal any significant intergroup differences during the study, whereas food consumption was reduced during the first days of treatment in all treated groups. This reduction in food intake was maintained at the high dose throughout the treatment period in both males and females but particularly for main phase females. This reduction in food consumption was considered to reflect a reluctance to eat the diet admixture due to its low palatability, particularly at the high dose. During the lactation period, the dietary intake of main phase females was reduced (up to 37% decrease compared to controls at 12000 ppm) in all test item treated groups. In main phase females, the overall reduction of food consumption throughout the study was 7, 11 and 23% of control group for 3000, 6000 and 12000 ppm groups, respectively.

In males in the 12000 ppm group, the reduction in bodyweight gain was particularly high during the first week of treatment (-80%) and remained lower than controls throughout the treatment period (-17% between days 8 - 42). During the recovery period, previously treated males at 12000 ppm showed a 50% increase in bodyweight gain compared to controls. During the pre-pairing period, females showed a decrease in bodyweight gain, especially during the first week of the treatment. From day 8 to the end of the study, toxicity group females showed similar mean bodyweight gain when compared to controls at 3000 and 6000 ppm but a 25% decrease in mean bodyweight gain compared to controls at 12000 ppm. During the recovery phase, their mean bodyweight gain was 3 times higher than the concurrent control group. During gestation period, main phase females showed an overall decrease in mean bodyweight gain at 3000, 6000 and 12000 ppm (-11, -6 and -33%, respectively), mainly observed during the last week of gestation. This decrease in bodyweight gains was also observed during lactation period (-50%) in all treated groups compared to controls.

No treatment related findings were observed in organ weights. Treatment with the test item at the dose levels of 12000 ppm caused a statistically significant decrease of total bilirubin, sodium level, globulin and tryglycerid, and increase in creatinin, ALP and albumin in males and decreased potassium level in recovery phase females. No further changes of biochemical blood parameters which were considered to be test item related were found in males or females at any dose level. Three main phase group males treated with 12000 ppm of the test substance revealed an enlarged liver that correlated with slight centrilobular hypertrophy of liver cells which was considered to be test item-related. Microscopic examination of liver sections revealed minimal centrilobular and partially reversible liver cell hypertrophy in all males. Furthermore treatment-related irritant effects were present in forestomach of main phase males and toxicity phase females.

No treatment related effects on mating, fertility and gestation length were observed. Therefore, the NOAEL concerning effects on fertility was set to the highest dose tested of 12000 ppm.

Effects on developmental toxicity

Description of key information

Developmental toxicity/teratogenicity
- NOAEL = 300 mg/kg bw/day (OECD 414, oral, test substance Geraniol Extra)

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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: 145.5–192.0 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 calibrated beaker and intensely mixed with a magnetic stirrer until it is 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:
30, 100 and 300 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

In a maternal toxicity rangefinding study dose levels of 300 an 1000 mg/kg bw/d were testet. The test substance was administered to pregnant female rats on GD 6 through GD 19. In this study a dose-related decrease of corrected body weight gain was noted, weight gain was approx. 9 and 12% below concurrent control, respectively. In addition increased relative weights of adrenals (116%), kidneys (117%) and liver (131%) were increased above concur-rent control. Thus dose levels of 30, 100 and 300 mg/kg bw/d were considered appropriate for the present definitive study.

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: at least once a day, or more often when clinical signs of toxicity were elicited (GD 0-20)

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gross pathology

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

Details on maternal toxic effects:

Maternal toxic effects:no 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 (300 mg/kg bw/d), nearly all (24 out of 25) females of the mid-dose group (100 mg/kg bw/d) and two females of the low-dose group (30 mg/kg bw/d) showed transient salivation during major parts of the treatment period. Salivation persisted in the respective animals only for some minutes after daily gavage dosing (i.e. up to 20 minutes) and was initially observed on GD 7.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected.

- FOOD CONSUMPTION:
The mean food consumption of the dams in all test groups was generally comparable to the concurrent control throughout the entire study period.

- BODY WEIGHT:
The mean body weights (BW) and the average body weight gains (BWC) of the low-, mid- and high-dose dams (30, 100, 300 mg/kg bw/d) were in general comparable to the controls throughout the entire study period. This includes the statistically significantly higher BW/BWC values in the low-dose group on GD 19 and 20 (BW), GD 17-19 and GD 6-19 (BWC).

- CORRECTED (NET) BODY WEIGHT GAIN:
The corrected body weight gain of test groups 1-3 (30, 100 and 300 mg/kg bw/d) revealed no difference of any biological relevance to the concurrent control group. Mean carcass weights remained also unaffected by the treatment.

- UTERUS WEIGHT:
No test-substance related findings.

- NECROPSY FINDINGS:
No test-substance related findings.

- REPRODUCTION DATA:
The conception rate was 100% in all test groups (0, 30, 100 and 300 mg/kg bw/d).
No test-substance related findings in conception rate, in the mean numbers of corpora lutea, implantation sites and viable fetuses or the number of resorptions and the value calculated for postimplantation loss.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no 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 fetus with multiple external malformations was recorded in test group 2 (100 mg/kg bw/d). The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and was comparable to the histori-cal control data.

- FETAL EXTERNAL VARIATIONS:
no external variations were recorded

- FETAL EXTERNAL UNCLASSIFIED OBSERVATIONS:
no unclassified external observations were recorded

- FETAL SOFT TISSUE MALFORMATIONS:
One soft tissue malformation was recorded for two control fetuses and one mid-dose fetus (0 and 300 mg/kg bw/d). The overall incidences of soft tissue malformations were comparable to those found in the historical control data.

- FETAL SOFT TISSUE VARIATIONS:
no soft tissue malformations were recorded

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

- FETAL SKELETAL MALFORMATIONS:
Two fetuses bearing skeletal malformations were detected in test groups 0 and 2 (0 and 100 mg/kg bw/d) affecting the skull, vertebral column (with or without involving the ribs) and humerus. An association of these malformations to the treatment is not assumed.

- 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 skeleton and appeared without a relation to dosing. The overall incidences of skeletal variations were comparable to the historical control data.

- 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 skull, the sternum and ribs and did not show any relation to dosing. The incidence of branched rib cartilage was significantly increased in test group 2 (100 mg/kg bw/d). However, this finding showed no dose-dependency and was therefore assessed to be without biological relevance

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at highest tested dose

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

Based on the results obtained in the study, the NOAEL for maternal toxicity and for developmental toxicity could be set at 225 mg/kg bw/day.

Executive summary:

The effects of the test item were investigated in rat during pregnancy and embryo-foetal development.

Females were mated with sexually mature males of the same strain and then assigned to 4 groups of 25 females each. All femaleswere administered by oral gavage during the gestation period fromDay 6 through Day 19 post coitum at 75, 225 and 750 mg/kg bw/day and 10 mL/kg body weight as the dose volume. Control females received the vehicle (0.5% carboxymethylcellulose in softened
water (0.5% CMC)) at the same dose volume during the same treatment period.

No mortality of females occurred during the study. The final number of pregnant females on Day 20 post coitum was 25 in the control group,low- (75 mg/kg bw/day) and top level (750 mg/kg bw/day) and 23 in the mid-dose group (225 mg/kg bw/day).

Piloerection was the treatment-related clinical sign noted in all females receiving 750 mg/kg bw/day. Other clinical sign, such as hunched posture was also noted. No other relevant clinical signs were noted in females receiving 75 and 225 mg/kg bw/day.

Reduction in maternal body weight (not greater than 10%) was observed at 750 mg/kg bw/day between Days 12 to 20 post coitum, when compared to the control group. The maternal growth retardation was better evident on body weight gain which was decreased from Days 9 to 20 post coitum, with significance, at statistical analysis, when compared to the control group.

Statistically significant reduction in food consumption was noted in the high-dose females receiving 750 mg/kg bw/day starting from Day 9 post coitum until termination. Some differences (decrease) observed in the remaining treated groups against to control, was transient and with a trend of recovery.

There was no treatment-related effect on thyroid hormone determination.

A decrease in body weight gain, corrected for gravid uterus weight, terminal body weight and body weight at Day 6 post coitum was recorded in treated females receiving 750 mg/kg bw/day (approximately 20%). Terminal body weight and corrected maternal body weight were also statistically significant decreased in females dosed at 750 mg/kg bw/day, when compared to the control group.

Litter data, mean foetal weight and sex ratios did not show relevant differences between control and treated groups.

Compared to the control, no alterations were seen in the anogenital distance performed in foetuses maternally exposed to the test item.

Statistically significant increase in absolute and relative mesenteric nodes weight was observed in females receiving the dose levels ≥ 225 mg/kg bw/day, when compared to the controls.

Females that completed the treatment period did not show relevant macroscopic changes.

No treatment-related changes were noted in thyroid when examined microscopically.

The overall incidences of foetuses or litters with findings did not indicate test item-related effect.

No relevant changes that could be considered treatment-related were observed at visceral examination of foetuses between the control and treated groups. Consequently, under the conditions described for this study, the test item did
not reveal teratogenic potential up to and including the dose level of 750mg/kg bw/day.

The skeletal assessment revealed instances of an increase incidence of irregular sternebral ossification in the high dose foetuses (750 mg/kg bw/day) compared to control. The alterations noted can be considered as a transient delay of ossification that will resolve shortly after birth rather than as teratogenicity sign.

Effect on developmental toxicity: via oral route

Dose descriptor:

NOAEL

300 mg/kg bw/day

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Developmental toxicity was evaluated in a study performed according to OECD Guideline 414 (BASF SE, 2016). The test substance Geraniol Extra was administered as a solution in corn oil to 25 "time-mated" (mated by breeder) female Wistar rats/group by stomach tube at doses of 30, 100 and 300 mg/kg bw on day 6 through day 19 post coitum (p.c.). On day 20 p.c., all females were sacrificed and assessed by gross pathology including the uterus and the placentae where corpora lutea were counted and number and distribution of implantation sites (differentiated as resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed, and further investigated for any external findings. Thereafter, one half of the fetuses of each litter 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 Geraniol Extra to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 300 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity.

In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 300 mg/kg bw/d.

 

In an OECD Guideline 443 (Charles River, 2021) doses of 50, 200 and 800 mg Geraniol/kg bw/d were administered to male and female F0 wistar rats from 10 weeks prior to mating continuously until termination, F1 animals from Post Natal Day 21 (except Cohort 2B, which was not dosed) continuously until termination via gavage.

This study was designed to provide an evaluation of the pre- and postnatal effects on development as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and their offspring. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, physical and functional development until adulthood, and nervous and immune system development was undertaken to identify specific target organs in the offspring. In addition, the study provided information about the effects of the test substance on the integrity and performance of the adult male and female reproductive systems.

For the F1 generation, the highest dose level of 800 mg/kg/day was reduced to 600, then 400 mg/kg/day due to intolerance (mortality) and titrated back via 600 to 800 mg/kg/day within the first two weeks of dosing (by Post Natal Day 33). This dose level is simply referred to as 800 mg/kg/day. Two F1 weanlings were euthanised on Day 1 or Day 2 of dosing due to clinical signs of intolerance to dosing at 800 or 600 mg/kg/day.

Compared to control animals:

Transient salivation and ploughing behaviour occurred in a dose-related manner in response to dosing in F0 and F1 animals at 800 and 200 mg/kg/day, and in F0 animals at 50 mg/kg/day.

Red paws (all) were noted for all F0 animals for several hours after dosing at 800 mg/kg/day on Day 8 only. Red ears were similarly noted for these animals and for most animals at 200 mg/kg/day between Days 8 and 10.

Treatment of F0 and F1 animals at 800 mg/kg/day was also associated with very low incidences/frequencies of transient post dose abnormal (uncoordinated) gait, decreased activity/subdued behaviour, low carriage, erected fur and chewing action. Some of these signs are consistent with the published observation that Geraniol is a CNS depressant (Medeiros et al 2018). Partially closed eyes, irregular respiration/breathing and pallor were also observed in F1 animals only. In addition, a small number of F1 animals at all test item dose levels had thinning fur/fur loss later on in the treatment period.

F1 animals at 800 mg/kg/day had slightly lower body weight values (up to 18% for males and 13% for females) during Weeks 1 and 2 of dosing, which corresponded with the time period during which intolerance and mortality occurred. Maternal weight gain from LD 1-21 was also lower: -12 and -24% at 200 and 800 mg/kg/day respectively.

At 800 mg/kg/day, slightly higher food consumption was noted for F0 females intermittently during the pre-mating period (up to 15%) and GD 14-20 (minimal at 9% but consistent with values up to 13% higher during GD 0-14). During lactation, intake was consistently slightly lower (by 10 to 16%), also for 200 mg/kg/day during LD 1-7 (by 10%).

At 800 mg/kg/day, slightly higher food consumption was recorded for Cohort 1B males from Week 16 and for Cohort 1A and 1B females between Weeks 6 and 13 (9-20%). Slightly higher food consumption was also noted throughout gestation (Cohort 1B): up to 16% at 200 and 800 mg/kg/day.

F0 pup survival between LD 0 and 4 was lower at 800 mg/kg/day (Viability Index 85.9%, with several litters losing 2 or 3 pups), with pup mortality occurring in 11 litters compared with 1 litter in each of the other groups, including control.

Some lower levels of motor activity were recorded for F1 males at 800 mg/kg/day (-15 to -16%, not statistically significant) and for females at all dose levels, in a dose-related manner: -12 to -19% ambulation only at 50 mg/kg/day (not statistically significant), -14 to -33% and -17 to -43% at 200 and 800 mg/kg/day respectively. These values also reflected changes seen in increasing numbers of individual sessions, from at least half at 50 mg/kg/day to most/all at 200 and 800 mg/kg/day.

In the F1 generation Functional Observation Battery (FOB), low incidences of severe salivation, with none in the control group, and uncoordinated landing on air righting at 800 mg/kg/day were considered to be related to the clinical observations at this dose level. A small difference in mean forelimb grip strength was noted for males: -17% at 50 and 200 mg/kg/day, -14% accompanied by -11% for hindlimb grip strength at 800 mg/kg/day (no statistical significance).

F1 generation coagulation, urinalysis, estrus cycles and mating performance, duration of gestation, litter survival and performance, anogenital distance, nipple retention, vaginal opening, balano-preputial separation, acoustic startle, gross pathology, neuropathology, spleen immunophenotyping, ovarian follicle counts. The test item did not suppress the IgM response.

For further details on maternal toxicity and fertility/reproductive performance please refer to the section "Toxicity to reproduction".

In terms of the study objectives, it was concluded that administration of Geraniol by daily oral gavage administration was not initially tolerated by F1 weanling rats at dose levels of greater than 400 mg/kg/day, being associated with mortality at 600 and 800 mg/kg/day. Target organ effects were primarily adaptive in nature, affecting liver, thyroid and spleen at dose levels of 800 mg/kg/day (F0 generation) and from 50 mg/kg/day (F1 generation). However, the olfactory epithelium degeneration at 800 mg/kg/day was an adverse change because it involved a neural tissue that does not regenerate. Based on the results, the following no-observed-adverse-effect levels (NOAELs) were assigned:

Systemic toxicity of adult rats = 200 mg/kg/day (based on olfactory epithelium degeneration)

Offspring viability, neonatal health, developmental status at birth = 200 mg/kg/day (F0 litters, based on pup survival) and 800 mg/kg/day (F1 litters)

Physical development until adulthood = 200 mg/kg/day (based on early mortality)

Pre and postnatal functional development until adulthood = 800 mg/kg/day

Nervous system and immune system development = 800 mg/kg/day.

Integrity and performance of adult reproductive systems = 800 mg/kg/day.

 

In a dermal screening study according to OECD 421 (BASF SE, 2010), 10 young male and female Wistar rats per dose (0, 50, 150 and 300 mg/kg bw/day) were treated daily with geraniol dissolved in corn oil for at least six hours on the clipped intact dorsal skin (semiocclusive dressing). About 2 weeks after the beginning of treatment, animals were mated to produce a litter. Mating pairs were from the same dose group. Treatment of the dams was discontinued from GD 20 onwards. Pregnant females were allowed to give birth and the offspring was brought up until postnatal day (PND) 4. The study was terminated with the sacrifice of the pups on PND 4 and of lactating dams shortly thereafter. The live birth indices as well as the rate of stillborn pups was comparable between all test groups and the control and reflected the normal range of biological variation inherent in this strain. None of the pups died during lactation in the control group and in all test groups. F1 pups did not show adverse clinical signs up to scheduled sacrifice. Therefore, also the NOAEL for developmental toxicity / teratogenicity is shown to be >300 mg/kg bw/day via the dermal route.

 

ADDITIONAL STUDIES

Apart from studies with the test substance geraniol, studies using structurally similar compounds were taken into account to address the endpoint of toxicity to reproduction. Geraniol (trans-3,7-dimethyl-2,6-octadien-1-ol) and nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) are trans/cis-isomers and therefore differ only in their relative orientation of functional groups within the molecule. Furthermore, relevant physicochemical parameters show comparability between geraniol and nerol.

 

Developmental toxicity was evaluated in a study performed according to OECD Guideline 414 (BASF SE, 2015). The test substance Geraniol 60 was administered as a solution in corn oil to 25 "time-mated" (mated by breeder) female Wistar rats/group by stomach tube at doses of 100, 300 and 1000 mg/kg bw on day 6 through day 19 post coitum (p.c.). On day 20 p.c., all females were sacrificed and assessed by gross pathology including the uterus and the placentae where corpora lutea were counted and number and distribution of implantation sites (differentiated as resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed, and further investigated for any external findings. Thereafter, nearly one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal findings.

As a result, administration of 1000 mg/kg bw elicited substance-induced effects on the dams including signs of maternal toxicity like reduced body weight gain on GD 6-8 (-53%) and GD 10-13 (-28%) resulting in about 10% less weight gain during treatment period (GD 6-19). The reduced corrected (net) body weight gain was about 14% below the concurrent control value. The dosage of 300 mg/kg bw/day resulted in reduced body weight gain on GD 6-8 (-43%) and reduced corrected (net) body weight gain about 13% below the concurrent control value. At the low dose (100 mg/kg bw/day) no substance-induced effects on dams occurred. The test substance had no influence on gestational parameters. Fetal examinations revealed that there is no effect of the compound on the respective morphological structures up to the highest dose tested (1000 mg/kg bw/d). Incidences of dilated renal pelvis and incomplete ossifications of various skeletal elements represent temporary delays in development which have no permanent effect on morphology and function of the affected organs or structures.

Based on these results, the no observed adverse effect level (NOAEL) for maternal toxicity is 100 mg/kg bw/day. The NOAEL for prenatal developmental toxicity could be fixed at 300 mg/kg bw/day.

 

The oral screening study according to OECD 421 with the reaction mass of geraniol and nerol (E- and Z-isomer) (BASF SE, 2010) showed effects on pup survival. The dose levels were 0, 100, 300 and 1000 mg/kg body weight/day. About 2 weeks after the beginning of treatment, animals were mated to produce a litter. Mating pairs were from the same dose group. Pregnant females were allowed to give birth and the offspring was brought up until postnatal day (PND) 4. The study was terminated with the sacrifice of the pups on PND 4 and of lactating dams shortly thereafter. Pregnancy was unaffected at the low-dose. However, there is an alert for a dose-dependent adverse effect of the test substance on pre-/postnatal development of the F1 offspring at mid and high-dose level (300 and 1000 mg/kg bw/d). For the high-dose a lower live birth index (89 %) was noted. Pup survival until PND 4 was decreased by 25% and average pup body weight on PND 4 was decreased by 18%. This reduced live birth index was due to losses in only one animal in the group (all others showed no losses). The significantly reduced postnatal offspring weight/weight gain during the first 4 days after birth are likely related to maternal toxicity and ability to care and nurse for the pups as evidenced by clinical observations, empty stomachs in 10% of pups and significantly reduced feed consumption and body weights during the lactation period. Overall, it may be considered that the pup effects seen are secondary to maternal toxicity which effects pup care and nursing. At the mid dose level, the same effects were noted, but at a lesser incidence and no significant effect on weight/weight gain was observed. The mid-dose also had a lower live birth index (94%). The slightly higher (non-significant) number of stillborns may well be contributed to the greater litter size in this group, which leaves the adverse effects on development of offspring in the mid-dose group to be limited to a slightly reduced pup survival (-9%). At least partially, the reduced pup survival may be secondary to a disturbance of maternal care as it became obvious by empty stomachs in pups which have been observed in 5% of mid-dose. In addition, findings in the mid dose appear to be limited to one animal. This animal appears to have maternal toxicity issues as evidenced by clinical observations (pups not properly nursed and insufficient maternal care of the pups), significantly reduced feed consumption and empty stomachs in the pups. These effects are similar to those seen at the top dose and it may therefore be concluded that maternal toxicity in this one animal is also responsible for the effects seen. The findings in this one animal appear to be an outlier in this group and excluding this animal from the data would show no significant findings vs. controls. No such findings were noted at the low-dose. Overall, maternal toxicity as evidence by lack of care of the pups, significantly reduced feed consumption in the dams and empty stomachs in the pups appears to be responsible for the findings seen at the top and mid dose groups. Effects are mainly due to one animal each at either dose group. The observed findings show a questionable correlation between the effects seen on maternal toxicity and effects seen on the offspring. Hence, their relevance based only on this screening study is doubtful and more complete studies at tolerated doses are required. Furthermore, it is unclear whether these effects are due to the corresponding amount of geraniol, which is 600 and 180 mg/kg bw/day in high and mid dose, or the respective amount of nerol, which is 400 and 120 mg/kg bw/day in high and mid dose. Therefore, this study cannot be regarded to assess the potential of geraniol alone and no NOAEL can be derived.

 

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (OECD TG 422) has been performed (DRT, 2013). The test item nerol was administered by dietary admixture (initially mixed with 2% corn oil) to three groups of Han Wistar rats for up to 42 consecutive days (main phase males, toxicity females and recovery animals) or between 41 and 53 days (including three weeks exposure phase, pairing, gestation and early lactation; main phase females) at dietary concentrations of 3000, 6000 and 12000 ppm (equivalent to 191.2, 374 and 720 mg/kg bw/day, respectively). A control group was treated with basal laboratory diet (with 2% corn oil). Two recovery groups, each of five males and five females, were treated with the high dose (12000 ppm) or basal laboratory diet alone for 42 consecutive days and then maintained without treatment for a further 14 days. Pairing of main phase animals and recovery males within each dose group was undertaken on a one male:one female basis within each treatment group on Day 22 of the study, with females subsequently being allowed to litter and rear their offspring to Day 7 of lactation.

Test-item related finding observed in parental animals are described in detail in the discussion on fertility effects and in IUCLID Chapter 7.8.1.

No treatment related effects were noted in the length of gestation between control and treated groups. Implantation sites were slightly reduced at 3000 and 12000 ppm, without reaching statistical significance). A significant dose-related increase in post-implantation loss was observed: control 5.6%, 3000 ppm 11.0%, 6000 ppm 13.5% and 12000 ppm 18.0% (mean values of mid and high dose animals outside the historical control data). This change was considered to be test item-related. Other parameters of offspring growth and developmental were not affected by the treatment with the test item. Under the chosen test conditions the NOAEL for developmental toxicity was set to 3000 ppm (corresponding to 191.2 mg/kg bw/day) due to the observed post-implantation losses. However maternal toxicity was even observed at the lowest concentration tested, namely decrease in bodyweight gain at 3000 ppm in females during the last week of gestation (-17%) and during early lactation (-67%, associated with -20% in food consumption). The LOAEL for maternal toxicity was set at 3000 ppm. Overall, maternal toxicity may be associated with the post-implantation losses observed in this study.

 

 

Justification for classification or non-classification

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