Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jun 2017 - Apr 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 29 July 2016
Deviations:
yes
Remarks:
Detailed deviations from the study plan are described in the "Principles of method if other than guideline" part
Qualifier:
equivalent or similar to
Guideline:
other: OECD 412
Version / remarks:
Principles of inhalation exposure were planned according to OECD 412
Deviations:
not specified
Principles of method if other than guideline:
The following deviations from the study plan occurred during the study but were not considered to have compromised the validity or integrity of the study:

1. Control formulations were not sampled for Weeks 1and 2 as stated in the original signed study plan. Control animals were exposed to air only and control formulations were not required for this study design.
2. The eyes from Animal No. 4501 were inadvertently placed in 10% NBF rather than Modified Davidson’s solution.
3. Animal Nos. 3008 and 3009 were exposed to the test item for approximately 4 hours and 36 minutes on Day 14 rather than 6 hours as per study plan. These animals were found in questionable health in the morning and it was determined that they were dehydrated due to poor positioning of the water valve. These two animals were allowed to drink water for at least 1 hour and then were placed into the exposure chamber.
4. On 28 August 2017, post-exposure observations and PM viability checks were not performed on any of the animals.
5. Twice daily parturition observations were not performed for animals on GD 18 (4 Sep 2017) due to a scheduling error. Morning parturition observations were not performed for animals on GD 18 and GD 19 (5 Sep 2017) for several animals due to a scheduling error.
6. Hormone serum samples for Animal Nos. 1001, 1002, 1004, 1005, 3001, 3002 and 4001 were transferred late which resulted in over-processing.
7. The thyroid hormone pooled blood sample from Litter 2502 was not allowed to clot for 30 minutes at room temperature due to a transcription error of 0913 on the sample tube. The sample was actually collected at 0931, centrifuged at 0949 and harvested and frozen at 1001.
8. Documentation was not made for the location (intended for the caudal area) of the implanted microchip in all animals.
9. Animal Nos. 1503 and 4504 were not fasted overnight prior to terminal necropsy or the terminal clinical pathology and thyroid hormone blood sample collection.
10. The head of Animal No. 4505 was not collected; therefore, the pituitary gland and the cervical vertebra were not retained and the pituitary gland could not be weighed.
11. On 9 and 10 October 2017 observations during lactation were not performed prior to exposure.
12. Samples collected on LD 14 (Animal Nos. 2502 and 2503) and PND 13 (Animal/Pup Nos. 2501-1, 1506-8 and 4509-3) were processed 1 to 10 minutes late. One sample on LD 14 (Animal No. 4505) was processed almost 2 hours late.
13. The physical observations animals were performed prior to the initiation of exposure rather than post-exposure on several occasions during the treatment, gestation and lactation phase.
14. On treatment Day 1 (3 August 2017), the pre-exposure observation occurred after exposure initiation for Groups 1 and 2. Exposure observations were not performed as follows:
Date Timepoint Animals Phase(s)
18, 19 Aug 2017 Pre-exposure All Mating and gestation
21, 27, 30, 31 Aug 2017 Pre-exposure All All phases
29 Aug 2017 Pre-exposure All Mating and treatment
13 Sep 2017 Pre-exposure All Mating
On 28 Aug 17, only pre-exposure observations were performed for females in offset 1.
15. The twelve hour light/dark cycle was interrupted on one occasion on 16 August 2017. The cycle was turned on 22 minutes after the lights were off in order to perform PM daily viability observations.
16. Vaginal smears were not collected at termination on LD 14 from Animal No. 2508 and Animal No. 4507.
17. Pre-exposure observations were performed late (during exposures from 1455 to 1456) for all animals in the mating and males in the post-mating phase on 5 September 2017. Animals were loaded into tubes for test item administration between 0912 and 0940 and exposures began shortly afterwards.
18. The lactation start date for Animal No. 3502 was incorrectly entered into the Pristima computer system. Due to computer limitations, the start date for this animal could not be corrected and data was collected; however, not for the correct lactation day. Lactation data for this animal is excluded from summary tables and calculation of mean values.
19. The method of euthanasia cannot be confirmed due to lack of documentation for pups from the litter of Dam No. 3501.
20. Authorization was not obtained from the Study Director to store the formulation samples for Weeks 2, 6, 7, 8 and 10. Aerosol samples from Group 1 were analyzed individually instead of combining into one sample for assay.
21. The nasal turbinates for Animal Nos. 1510, 2507, 4501 and 4504 were inadvertently stored in the wrong cassettes or labeled incorrectly in the blocks. As a result, the turbinates for these animals could not be properly identified and are not being reported.
22. Animal No. 4504 was inadvertently omitted from the rotation pattern on the animal rotation/sampling location form on Exposure Days 43 (LD 7) and 44 (LD 8); therefore, dosing could not be confirmed for those two days for that animal.
23. Week 1 particle size determination samples were not collected for Groups 2 and 3. This was a planned deviation.
24. The following tissues were missing or inadequate at the time of microscopic evaluation for the animals outlined below:
Tissue Animal No(s).
Eyes 4003
Parathyroids 1504, 2507
Pharynx, nasal 4501, 4504, 2507, 1510
Pituitary 4505
Spinal Cord, cervical 4505
Tracheal Bifurcation 4003
25. Physical examinations were inadvertently performed on all females in Offset 1 on Mating Day 1 (17 Aug 2017) and on Female Nos. 2502, 2503, 2507, 2509, 3502, 4506 and 4507 on Mating Day 8 (7 Sep 17).
26. Positive signs of mating were missed for Female No. 2509; therefore, a physical examination, body weight and food consumption data were not collected for GD 0 and GD 7. After review of the vaginal smear data on 13 Sep 2017, she was moved to a gestation start date on 3 Sep 2017. Data for this female was being collected weekly and body weight and physical examination data performed on 7 Sep 2017 was collected while she was still in the mating phase on Day 8. This female was not pregnant.
27. On 31 Aug 2017, a physical examination was not performed for offset 2 males (mating Day 1) and offset 1 males (post-mating Day 1).
28. Female No. 4503 failed to litter and was euthanized on GD 26 instead of GD25.
29. Positive signs of mating were missed for Animal No. 2503 during the first phase of the cohabitation period. A weekly body weight was not taken for this female during the second phase of the cohabitation period.
30. The Study Plan indicated that T4 analysis would take precedence if there was a shortage of serum from any animal. For some animals, the T4 result is indicated to be QNS yet there is a result for TSH.
31. The Study Plan states that females placed in the study groups will have demonstrated 4 to 5 days of cyclicity during the pretest period. Some females placed on test had 3 days cyclicity and Study Director did not approve the use these animals.
32. Statistical Analysis of motor activity data was not included in the Study Plan yet it was performed.
33. The pretest period was 3 weeks while section 6.1.1 of the Study Plan required approximately 2 weeks.
34. The Study Plan indicates that assessment of thyroid hormones from the F0 females and PND 4 pups would be done if requested by the Study Director and after consultation with the Sponsor. These samples were analyzed but the Sponsor was not consulted and the Study Director did not make this request.
35. The following could not be confirmed because the proper documentation could not be located:
• Tattooing of the following dams and litters: 3503 on 11 Sep 2017, 4504 on 8 Sep 2017 and 4506 on 1 Oct 2017.
• Method of euthanasia (IP injection of sodium pentobarbital) for pups from Dam Nos. 3506 and 3507.
• Method of euthanasia (Isoflurane inhalation followed by exsanguination) for Animal No. 3508.
36. Several blood samples for hormone analysis at termination, PND 4 and PND 13 were not allowed to clot for 30 minutes (ranging from 18 to 29 minutes early) or harvested within 1 hour (ranging from 4 to 30 minutes late).
37. Observations of the thyroid with parathyroid gland, tracheal bifurcation, larynx, nasal turbinates, tracheobronchial lymph node and nasal pharynx for Animal Nos. 3502 and 3504 were not performed at the time of necropsy because the Pristima protocol was not updated to reflect the changes made in Study Plan Amendment No. 4.
38. The thyroid glands were inadvertently collected, weighed and sent to histology from unscheduled decedent Animal Nos. 3504-5M and 3504-12F.
39. Due to the small size of the pups and/or difficulty obtaining a blood sample, the blood volume was less than 0.6 mL from the following pups at PND 4 (pooled sample) and PND 13:
Litter: 4504, 1503, 1501, 1502, 2505, 2503, 2505, 4505, 1510 and 4510
Pups: 2510-10F, 4510-15F, 4510-16F, 3509-6M, 3509-7M, 3509-11F, 3509-12F and 2508-10F
40. The study plan states that the day on which parturition is complete will be defined as LD 1; however, several animals had parturition finish in the afternoon and LD 1 was documented as the next day which follows the company SOP (TX-05-1.4.1).
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
[Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
- Age: Approximately 11 to 12 weeks
- Weight (Day P21): Males=354 g to 451 g, Females=209 g to 291 g
-Light/dark cycle: A twelve hour light/dark cycle was provided and controlled via an automatic timer.
Note: Light cycles were interrupted on days when functional assessments, motor activity or fasting was conducted as required.
-Temperature and relative humidity: Monitored and maintained within the range of 20 to 26 °C and 30 to 70%, respectively.
Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration, had no effect on the daily average and were not considered to have influenced the health of the animals and/or the outcome of the study.
-Cages: Polycarbonate cages with a stainless steel mesh lid.
-Number of animals per cage: From arrival until one day prior to treatment, animals were pair or group housed (2 or 3 rats of the same sex per cage, respectively) in solid bottom cages with cellulose-based contact bedding.
From the initiation of exposure (pre-cohabitation phase), the F0 males and females for each set were pair-housed [same sex and treatment group per cage (except during the cohabitation phase)]. All F0 females were housed in suspended, solid bottom cages with cellulose-based contact bedding. Per the OECD 422 guideline, the F0 females were individually housed during presumed gestation and housed with her litter after delivery.
During cohabitation, the male and female rats were co-housed (1:1) within each treatment group. Once mated, the female rat was removed from the mating cage and housed individually.
-Bedding: Alpha-Dri® cellulose-based contact bedding (Shepherd Specialty Papers, Kalamazoo, Michigan); Teklad 7070C Certified Diamond Dry Cellulose Bedding (Envigo, Madison, Wisconsin). Provided to each cage throughout the study and changed at appropriate intervals each week. Analytical results of the bedding, provided by the manufacturer, are maintained on file at the Testing Facility. There were no known contaminants in the bedding that were expected to interfere with the objectives of this study.
-Environmental enrichment: Provided to each cage throughout the study, except during lactation when huts were removed, and replaced when necessary.

Feed
-Diet: Teklad Global 16% Protein Rodent Diet (Certified), 2016C (Envigo, Madison, Wisconsin)
-Availability: Without restriction. Animals did not have access to food during the exposure period. Fresh feed was presented weekly in the home cage of each animal.
-Analysis: Analysis of each feed lot used during this study was performed by the manufacturer. Results were provided to the Testing Facility and are maintained on file at the Testing Facility. There were no known contaminants in the feed that were expected to interfere with the results of this study.

Water
-Supply: Potable water from the public supply (New Jersey-American Water Company, Cherry Hill, New Jersey) via an automated watering system.
-Availability: Without restriction. Animals did not have access to water during the exposure period.
-Analysis: Water analyses are conducted by New Jersey-American Water Company, Cherry Hill, New Jersey (Raritan-Millstone Plant) to ensure that water meets standards specified under the EPA Federal Safe Drinking Water Act Regulations (40 CFR Part 141). In addition, water samples are collected biannually from representative rooms in the Testing Facility; chemical and microbiological water analyses are conducted on these samples by a subcontract laboratory. Results of all water analyses are maintained on file at the Testing Facility. There were no known contaminants in the water which were expected to interfere with the results of this study.

Animals were monitored by the technical staff for any conditions requiring possible veterinary care. Miscellaneous, non-test item-related veterinarian evaluations for individual animals were reviewed by the study director and are documented in the study file.

Assignment and Identification

-Assignment: More animals than required for the study were purchased and stabilized.
Males considered suitable for study on the basis of pretest physical examinations, body weight data and any other pretest evaluations, were randomly assigned to control, exposure or spare groups, using a computerized program, in an attempt to equalize mean group body weights. Individual weights of male animals placed on test were within 20% of the mean weight.
Females considered suitable for study on the basis of pretest physical examinations, body weight data, regular estrous cycles and any other pretest evaluations, were randomly assigned to control, exposure or spare groups, using a computerized program, in an attempt to equalize mean group body weights. Normally cycling F0 females (demonstrating 4 to 5 days cyclicity) were selected for each exposure group. Individual weights of female animals placed on test were within 20% of the mean weight.

-Identification of animals: Each rat was implanted with a BMDS IMI-1000 Implantable Radio Frequency Transponder (microchip) programmed with a unique number. This number was cross referenced with an animal number assigned by the Testing Facility; this number plus the study number comprised a unique identification number for each animal. In addition, each cage was provided with a cage card that was color-coded for exposure level identification and contained study number and facility-assigned animal number information.
Each pup within a litter was assigned an identification number on PND 1 that was tattooed on the paws prior to the first body weight. This number, the dam number, and the study number comprised the unique animal number for each pup and were included on the cage cards.

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
nose only
Remarks on MMAD:
not applicable
Vehicle:
water
Remarks:
Tap water supplied by New Jersey-American Water Company, Cherry Hill, New Jersey, was distilled at the Testing Facility and stored at room temperature.
Details on exposure:
Please see "Any other information on materials and methods incl. tables"
Details on mating procedure:
Within each treatment group, the F0 male and female rats were co-housed (1:1 in the male’s cage) until evidence of mating was seen or until 14 consecutive days of cohabitation had elapsed. Female rats were observed each morning for the presence of a vaginal plug or sperm in the vaginal smear. If not mated, the stage of the estrous cycle was recorded (between 06:00 am and 10:00 am). The day on which evidence of mating was observed was defined as Day 0 of presumed gestation (GD 0). Once mated, the female rat was removed from the mating cage and housed individually until delivery. Presumed pregnant dams that did not deliver litters were euthanized on presumed GD 25 or 26.
After 14 consecutive days of cohabitation with no positive signs of mating, the pairing (with identified male and female) was deemed “unsuccessful” upon review of the estrous cycle for that female. The female was placed with a second suitable co-group male that had already successfully mated. The female remained with this second male until positive evidence of mating was detected or for a maximum of 7 days.
In the absence of positive signs of mating during the second pairing, the morning of the last day of cohabitation was considered “presumed GD 0” for that female. If the presumed pregnant dam did not appear visibly/palpably pregnant, she was euthanized on presumed GD 25. If a female showed no positive evidence of mating, but appeared visibly/palpably pregnant, it was allowed to deliver, and the estimated GD 0 calculated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
-Homogeneity: The test item mixtures are true solutions; therefore, no homogeneity analysis was performed. Documentation of formulation solubility is maintained by the Sponsor.
-Stability: Stability under storage conditions used in this study was determined from the exposure formulations prepared in validation Study DL10DX using the proposed preparation procedure for at least 8 days from preparation.
-Exposure Concentration: Two samples (2.5 mL each) were taken from the middle region of each formulation for Groups 2 to 4 for each week on the day of exposure preparation. One sample was analyzed, in duplicate, for exposure confirmation analysis and one sample was retained with a layer of nitrogen, light protected and refrigerated (2 to 8 °C) (retained sample was discarded after valid analytical results were obtained).
-Sample Storage Conditions: Exposure formulation samples were maintained with a layer of nitrogen, protected from light, and refrigerated (2 to 8 °C) until analysis.
-Method of Analysis: Analyses were performed by the Department of Formulation and Inhalation Analysis at the Testing Facility.
Duration of treatment / exposure:
6 hours/day

Frequency of treatment:
7 days/week
Details on study schedule:
The animals on study were acclimated to the method of restraint, over a 3 day period (a maximum of 10 minutes on Day 1, a maximum of 30 minutes on Day 2 and a maximum of 60 minutes on Day 3) immediately preceding the first test item exposure.

The duration of exposure for the F0 males was 2-weeks pre cohabitation, during cohabitation (up to 2 weeks) and continuing during post-cohabitation until the day prior to termination [approximately 35 days].
The duration of exposure for the F0 females was 2-weeks pre cohabitation, cohabitation (up to 3 weeks) and during gestation and lactation (GD 0 through GD 19 for females that delivered a litter or GD 25 or GD 26 for dams that did not deliver a litter) and LD 5 through LD 13 (approximately 70 days).

Restraint Devices
Animals were restrained (individual exposure tubes) during the exposure administration procedures for a maximum of 420 minutes per day. This daily period of restraint included the 360 minutes of exposure during which there were interruptions for technical reasons. This daily period of restraint also included the pre-exposure and post-exposure intervals in which the animals were placed in or removed from the exposure tubes. While the loading and unloading process for each animal only took a few minutes, the animals were initiated and terminated for exposure as a group and thus the pre-exposure and post-exposure duration in the exposure tubes was extended.
Doses / concentrationsopen allclose all
Dose / conc.:
10 other: µg/L
Dose / conc.:
15 other: µg/L
Dose / conc.:
20 other: µg/L
Dose / conc.:
0 other: µg/L
Remarks:
Control group: Air only
No. of animals per sex per dose:
Number of animals Received Placed on test
Total: 94 80
Males: 42 40
Females: 52 40

The study consisted of 1 control and 3 treated groups. 10 males and 10 females per group were applied.
Control animals:
yes, sham-exposed
Details on study design:
Please see "Any other information on materials and method inkl. tables"
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
Daily Observations
Animals were observed in their cages at least twice daily for mortality and general condition. Animals in extremely poor health or of questionable viability were identified for further monitoring and possible euthanasia.

Dosing Observations
During the treatment period, all animals were observed for signs of toxic or pharmacologic effects once prior to and once within 2 hours after test item administration.
Animals were observed on a group basis once during each exposure. Only animals with findings were reported.

Signs
F0 male rats were observed twice prior to initiation of dosing and once weekly (post-exposure on an exposure day) during the treatment period until termination.
F0 female rats were observed twice prior to initiation of dosing, once weekly (post-exposure on an exposure day) during the pre-cohabitation and cohabitation phases. Once mating was confirmed, animals were observed on GDs 0, 7, 14 and 20 and LDs 1, 4, 7 and 13. Maternal behavior was observed daily.
Examinations included observations of general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as evaluation of respiration.

Body Weight
Pretest body weights of all animals were recorded weekly prior to initiation of exposure and on the day prior to exposure initiation.
Body weights of the F0 male rats were recorded from the initiation of exposure of each group and at least weekly thereafter throughout the study until euthanized and at termination.
Body weights of the F0 female rats were recorded from the initiation of exposure of each group and at least weekly during the pre-cohabitation and cohabitation phases until mated. Mated female rats were weighed on GD 0, 7, 14 and 20 and female rats that delivered litters were weighed on LD 1, 4, 7 and 13 and at termination on Day 14.
Body weights were taken pre-exposure on an exposure day for all animals.

Food Consumption
Food consumption for the male and female rats was measured (weighed) weekly from approximately one week prior to initiation of exposure of each group (pretest) and weekly during the pre-cohabitation phase.
Food consumption was not measured during the cohabitation phase when male rats were being co-housed with female rats.
For male rats, food consumption was measured weekly (Days 1-7) during the post-cohabitation phase. For female rats, gestation and lactation food consumption was measured on GDs 0-7, 7 14 and 14-20 and on LDs 1-7 and 7-13, respectively.

Parturition and Lactation
On GD 18, several days prior to expected parturition, examination for signs of parturition was performed twice daily (morning and afternoon). Whenever possible a female that was in the process of delivery was not disturbed. Dosing and other activities (e.g., detailed physical observations and body weights) were not performed during parturition. The day on which parturition was observed as completed was defined as LD 1 (dams) and is equivalent to PND 1 (pups).

Functional Assessments
Sensory reactivity, grip strength and locomotor activity were performed post-exposure early in Week 5 for the first five F0 males in each group and on LD 8 ± 1 for the first five F0 females in each group as described below. Evaluations were not performed blind.

Sensory Reactivity
Sensory reactivity evaluations were performed for visual approach, audition assessment, pinna reflex, proprioception, pain perception, pupil response and air righting. Open field evaluations for abnormalities of posture, gait, vocalizations and any abnormal behavior were also performed.

Grip Strength
Forelimb and hindlimb grip strength evaluations were performed using a Chatillon Digital Force Gauge (Model E-DFE-010) and accompanying Columbus Instruments Grip Strength Platform.

Locomotor Activity
Spontaneous exploratory activity was measured. Each animal was placed in a separate shoebox cage in an automated Motor Monitor System (Kinder Scientific). Activity was monitored during a 60 minute session composed of 12, 5 minute intervals. The total number of horizontal and vertical photo-beam breaks that occurred during each of the 5 minute intervals was recorded.

Evaluations were performed according to the Testing Facility’s Standard Operating Procedures that include defined scales as listed below:

Reflex assessments

Visual Approach
(Visual Appr)
1 No reaction
2 Slowly approaches, sniffs and/or turns away
3 Freezes or pulls away slightly
4 Jumps or turns abruptly to avoid
5 Attacks and/or bites

Audition Assessment (Aud)
1 No reaction
2 Slight reaction, some evidence that noise was heard
3 Flinches and/or flicks ears
4 Exaggerated; jumps, flips, bites

Pinna Reflex
(Pinna Reflex)
1 Ear flattens against head or shakes head
2 No response

Proprioception (Proprio)
1 Returns leg to original position
2 Returns leg only partially to original position
3 No response; allows leg to remain pulled back

Pain Perception (Pain)
1 No reaction
2 Turns toward site, walks forward, or vocalizes with little or no
movement
3 Rat flinches, muscle contractions present
4 Exaggerated; jumps, bites, attacks

Pupil Response
(Pupil Resp)
1 Pupil constricts normally, pupil size not abnormal
2 Pupil size does not change
3 Miosis (right, left or bilateral indicated)
4 Mydriasis (right, left or bilateral indicated)

Air Righting (Air Right)
1 Normal; lands on four feet
2 Slightly uncoordinated; two-legged or unsteady landing
3 Lands on side
4 Lands on back

Other
Grip Strength Forelimb and hind limb grip strength measured in grams

Open field
Posture (Post)
1 Normal
2 Lying on side
3 Flattened
4 Crouched or hunched

Gait
1 Normal
2 Ataxia
3 Forelimbs dragging
4 Hindlimbs dragging
5 Forelimbs splayed
6 Body drags

Vocalizations (Vocal)
1 None
2 Present in open field
3 Present when handled and in open field
4 Present when handled

Abnormal Behavior
1 None present
2 Slight
3 Severe

Clinical Pathology
Blood was obtained from anesthetized (isoflurane) F0 rats as a terminal procedure via the vena cava from up to the first 5 surviving animals/sex/group. Animals were fasted overnight prior to collection.

Hematology
Blood samples (approximately 0.25 mL) were collected into tubes containing K2EDTA anticoagulant and analyzed for the following using a Siemens ADVIA 120 Hematology Analyzer:
Hemoglobin (HGB)
Hematocrit (HCT)
Red blood cell count (RBC)
Platelet count (PLT)
Mean corpuscular volume (MCV)
Mean corpuscular hemoglobin (MCH)
Mean corpuscular hemoglobin concentration (MCHC)
Red cell distribution width (RDW)
Total white blood cell count (WBC)
Reticulocyte count (RETIC)
Differential white blood cell count
Neutrophils (ANEU)
Lymphocytes (ALYM)
Eosinophils (AEOS)
Basophils (ABASO)
Monocytes (AMONO)
Large unstained cells (ALUC)

A peripheral blood smear was prepared for each animal at each blood collection interval and was available for confirmation of automated results and/or other evaluations deemed necessary by the Clinical Pathologist.
Oestrous cyclicity (parental animals):
During pretest (to determine suitability for study), pre-cohabitation and cohabitation phases, vaginal smears were obtained daily from each F0 female and the stage of estrous was determined. In addition, a vaginal smear was collected from each F0 female on the day of scheduled termination and evaluated for stage of estrous cycle.
Sperm parameters (parental animals):
Not determined.
Litter observations:
Litters were observed as soon as possible after completion of parturition for the number of live and dead pups, runts and pup abnormalities and the sex of each pup. Thereafter, litters were observed twice daily (morning and afternoon); all pups in the litter were uniquely identified by tattoo after parturition was completed. Because the litters were not culled, the full number of pups remained in each litter. The presence of dead pups was recorded and these were removed from the litter as found and necropsied.
Postmortem examinations (parental animals):
Necropsy

Macroscopic Examinations
Macroscopic postmortem examinations were performed on all F0 rats. Postmortem examinations included an external examination as well as a detailed internal examination. Special attention was paid to the organs of the reproductive system. The number of implantation sites, scars and corpora lutea was recorded for each female rat.
Macroscopic post-mortem examinations (external only) were performed on all surviving F1 pups on PND 13. One F1 male and one F1 female pup were randomly selected from each litter; the thyroid gland was removed, fixed, weighed, stored in formalin and processed for histopathology. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Unusual observations, including gross abnormalities were noted and then the carcasses were discarded.
F1 pups that died during the study were given a macroscopic postmortem examination. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Unusual observations, including gross abnormalities and the absence of milk in the stomach was noted. All carcasses were discarded following examination.

Scheduled Necropsy
Necropsy was performed on 10 F0 males/group after males had been treated for 5 weeks. Necropsy schedules were established to ensure that approximately equal numbers of males from each group were examined at similar times of the day.
Necropsy was performed on up to 10 F0 females/group on LD 14, after 50 to 59 days of exposure.
Female rats that failed to deliver a litter were euthanized on GD 25 or GD 26.

Method of Euthanasia
Adult males and females were euthanized by exsanguination following isoflurane inhalation.
Pups were euthanized using an intraperitoneal injection of sodium pentobarbital.

Organ Weights
From the animals surviving to term and that delivered a litter, up to 5 animals/sex/group had organs in Table 1 weighed and from the remainder up to 5 animals/sex/group had organs from Text Table 2 weighed. Organs were not weighed for any female that failed to deliver a litter. Prior to weighing, the organs were carefully dissected and properly trimmed to remove adipose and other contiguous tissues in a uniform manner. Organs were weighed as soon as possible after dissection in order to avoid drying. Paired reproductive organs were weighed separately; all other paired organs were weighed together.

The thyroid with parathyroid gland was weighed from one male and one female F1 pup from each litter.

Preservatives
Eyes and testes were placed in Modified Davidson’s solution initially and then retained in 10% neutral buffered formalin (NBF). Lungs were infused with 10% NBF prior to their immersion into a larger volume of the same fixative. All other tissues were preserved in 10% NBF.

Processing
After fixation, the tissues and organs from all animals were routinely processed, embedded in paraffin, sectioned at approximately 5 microns, mounted on glass slides, stained with hematoxylin and eosin and examined by light microscopy.
After decalcification the nasal turbinates were sectioned transversely following Histology Department guides. All sections were examined, post-fixation, for the presence of macroscopically visible morphologic abnormalities.
Two levels of the larynx were obtained following Histology Department guides.
Postmortem examinations (offspring):
Physical Examinations
Each pup was given a physical examination daily from PND 1 through PND 13. Observations of dead or missing pups were noted.

Body Weight and Sexing
Individual pup body weights were recorded on PND 1, 4, 7, 11 and 13. The sex of each pup was identified on PND 1 and verified on PND 4, 7, 11 and 13.

Anogenital Distance
Anogenital distance was measured in millimeters using a caliper for all pups on PND 4.

Nipple Retention
Nipple retention was assessed for all F1 male pups on PND 13.
Statistics:
All statistical analyses were carried out separately for males and females using the individual animal (or litter) as the basic experimental unit.
For litter findings, the litter was the treated experimental unit and the basis for statistical analysis and biological significance were assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.
Animals that were not pregnant were excluded from group mean calculations. Animal Nos. 2502 and 2503 had no evidence of mating (presence of a vaginal plug or sperm in the vaginal smear) and were excluded from calculation of group means during mating and gestation.
The following data types were analyzed at each timepoint separately:
-body weight
-body weight changes
-food consumption
-hematology
-coagulation
-clinical chemistry
-organ weights, organ/body and organ/brain weight ratios
-gestation length
-number of implantation sites and corpora lutea
-pre- and post-implantation loss
-F1 pup weights (each weighing interval during lactation)
-number of pups per litter
-sex ratio (% male pups per litter)
-ano-genital distance (pup as experimental unit, but taking litter size into account)
-litter survival indices
-mean pup survival indices (PND 1, 4, 7 and 13)

The parameters to analyze were identified as continuous, discrete or binary. Test-article treated groups were then compared to the control.
For further information on statistics please see "Any other information on materials and methods inkl. tables".
Reproductive indices:
Please see "Any other information on materials and method inkl. tables"
Offspring viability indices:
Please see "Any other information on materials and method inkl. tables"

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Nicotine exposure at 15 and 20 µg/L was associated with adverse clinical signs (including tremors, cold to touch after exposure and decreased activity) and mortality as discussed above. Tremors were seen as early as the first day of exposures but were only seen in females and were seen in somewhat greater prevalence at the 15 µg/L exposure level (as was mortality) than at the 20 µg/L exposure level. Males, also at the 15 and 20 µg/L exposure level, were seen with decreased activity and hunched posture.
Nicotine exposure at 10 µg/L was associated with substantially fewer adverse clinical signs but a single mortality as discussed above.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Test item-associated deaths occurred in females (only) at ≥ 10 µg/L and are summarized as follows:

Group Exposure Level (µg/L) Mortality in Males Mortality in Females
1 0 0 0
2 10 0 1
3 15 0 8
4 20 0 1

Tremors, consistent with the mechanism of action of the test item, occurred in the unscheduled decedents at ≥ 15 µg/L. Five females at 15 µg/L (Animals Nos. 3501, 3504 3505, 3508 and 3510) and 1 female at 20 µg/L (Animal No. 4501) were euthanized between Treatment Day 4 and LD 14 for welfare reasons of tremors and the major factor contributing to death was considered tremors. Three other females at 15 µg/L (Animal Nos. 3506, 3507 and 3502) were euthanized between LD 10 and 12 for other test item–related welfare reasons but all had clinical findings that included tremors. Animal Nos. 3506 and 3507 were euthanized on LD 10 and 12, respectively, due to poor clinical condition. The animals had no macroscopic findings and there were no microscopic findings indicative of a cause of poor clinical condition. The major factor contributing to death was considered to be test item-related poor body condition. Animal No. 2507 (at 10 µg/L) was euthanized on GD 23 due to poor clinical condition. There were no macroscopic or microscopic findings indicative of a cause of poor clinical condition and the major factor contributing to death was considered to be test item-related poor body condition.
Animal No. 3502 was euthanized on LD 10 due to skin ulceration/abrasion. Relevant macroscopic and microscopic findings included epidermal ulceration with inflammation and intralesional bacteria. The major factor contributing to death was skin lesions that were unlikely to have been directly related to the test item.
All of the unscheduled decedents (at all exposure levels) had microscopic findings consistent with chronic stress. Stress-related findings included adrenal cortical hypertrophy, thymic atrophy and/or uterine atrophy. Most unscheduled decedents at 15 µg/L (but not the decedent at 20 µg/L) also had minimal to marked hydropic degeneration and/or hepatocellular necrosis in the liver.
One female each in the control, 10 and 20 µg/L groups was euthanized due to failure to litter.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decreased body weights or body weight gains were seen during the pre-cohabitation period (up to 5% compared to controls) and mating/post-mating periods (up to 9% compared to controls) but only in the males at all exposure levels.
Decreased body weights or body weight gains were seen during the gestation period (up to 7% compared to controls) and lactation period (up to 3% compared to controls) in the females at all exposure levels.
In general, these adverse test item effects were similar at all exposure levels.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related effects on food consumption during the pre-cohabitation, cohabitation and post-cohabitation periods in the males and the pre-cohabitation, cohabitation and gestation periods in the females.
Decreased food consumption was seen during the lactation period (up to 32% compared to controls) in the females at all exposure levels. In general, these adverse test item effects were similar at all exposure levels.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Nicotine-related hematology changes at all exposure levels included increases in monocytes (2.32 to 3.91× control; females only). These changes were considered non-adverse due to their relatively small magnitude.
Animal no. 2505 had moderately lower platelet count that was considered not test item-related as it was a single incidence and lacked an exposure relationship.
All other differences between control and treated mean values, including those that were statistically significant, were considered not Nicotine-related as they lacked an exposure relationship and/or there was general overlap between individual control and treated values.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Nicotine-related clinical chemistry changes at all exposure levels included increases in alkaline phosphatase activity (1.3 to 5.13× control; at 10 µg/L, females only) and decreases in total cholesterol (0.61 to 0.68× control; females only) and triglycerides (0.46 to 0.59× control; females only). At ≥ 15 µg/L, increases in alanine aminotransferase activity (ALT; 1.46 to 2.00× control; males only) were considered Nicotine related. The increases in ALT correlated with hepatocellular necrosis in the liver of terminal necropsy males at ≥ 15 µg/L. These changes were considered nonadverse due to their relatively small magnitude and the decreases in total cholesterol and triglycerides in females were associated with decreases in food consumption.
All other differences between control and treated mean values, including those that were statistically significant, were considered not Nicotine related as they lacked an exposure relationship and/or there was general overlap between individual control and treated values.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no FOB observations that were considered test item-related.

Motor Activity
There were test item-related effects on horizontal and vertical movements in males during Week 5 and females on LD 8 at all exposure levels. However, any test item-related effects were transient (not seen at all intervals of measure) and all treated groups manifested normal habituation during the assay thus these findings were not considered adverse.

Horizontal
For males during Week 5, the 10 µg/L Nicotine treated group had significantly increased motor activity compared to control during interval 1 (p=0.001, Dunnett’s test). The 10, 15 and 20 µg/L Nicotine treated groups had significantly increased motor activity compared to control during intervals 2 and 3 (p≤0.014, Williams’ test). The 10 and 15 µg/L Nicotine treated groups had significantly increased motor activity compared to control during interval 4 (p≤0.021, Dunnett’s test). The 20 µg/L Nicotine treated group had significantly increased motor activity compared to control during interval 8 (p=0.047, Shirley’s test) and during interval 9 (p=0.040, Williams’ test).
For females on LD8, the 10, 15 and 20 µg/L Nicotine treated groups had significantly decreased motor activity compared to control during interval 1 (p≤0.009, Williams’ test). The Nicotine at 20 µg/L group had significantly decreased motor activity compared to control during interval 4 (p=0.047, Williams’ test).

Vertical
For males during Week 5, the 10, 15 and 20 µg/L Nicotine treated groups had significantly increased motor activity compared to control during interval 1 (p=0.001, Williams’ test).
For females on LD8, the 10, 15 and 20 µg/L Nicotine treated groups had significantly decreased motor activity compared to control during intervals 1 and 2 (p≤0.024, Williams’ test).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no organs weight changes directly related to the test item. Stress-related changes considered to be secondary to test item exposure occurred in the adrenal glands, spleen, thymus, prostate, uterus/cervix and pituitary gland at 10 ≥ µg/L.
Nicotine-exposed groups at ≥ 10 µg/L had higher mean adrenal and pituitary gland weights (absolute and relative to brain and body weight) and lower spleen, thymus, prostate and uterus/cervix gland weights (absolute and relative to brain and body weight) in one or both sexes compared to the control values. Higher adrenal gland weights and lower thymus and uterine weights corresponded microscopically to adrenal gland cortical hypertrophy, thymus atrophy and uterus atrophy respectively. The combination of organs weight changes and microscopic findings were consistent with chronic stress. For details on organ weight changes please see table 1 in "Any other information on results incl. tables"
All other organ weight changes, including those that were statistically significant, were considered not to be test item-related because they were sporadic, independent of exposure level, small in magnitude, occurred only in one sex and/or did not have macroscopic or microscopic correlates.

Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Pale areas in the liver of 1 terminal necropsy female (Animal No. 2504) correlated microscopically with slight hepatocellular necrosis (discussed below).
All other macroscopic findings occurred sporadically without a relationship to exposure level, were typical of background findings seen in this species or occurred at a similar incidence in control and test item-exposed groups. They were considered incidental and not related to test item exposure.

For further information please see "Terminal Necropsy" in section "Any other information on results incl. tables"
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Test item-related microscopic findings were present in the liver at ≥ 15 µg/L. Secondary changes due to the test item-associated chronic stress were present the adrenal gland, thymus and uterus at ≥ 10 µg/L.
For further information please see "Terminal Necropsy" in section "Any other information on results incl. tables"
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
please see "Any other information on results incl. tables"

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
There were test item-related effects on estrous cycling at all exposure levels. Specifically, the percentages of irregular cycles ranged from 75 to 100% for the test item-treated females. The mean cycle lengths increased for all test item-treated females from 2.1x to 2.8x control.
Despite the test item-related effect on estrous cycle number and duration, there was no impact on mating and fertility and therefore these effects were considered non-adverse.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no test item-related effects on mating and fertility. Furthermore, there were no test item-related effects on the number of implantation sites, gestation length, total litter size, or live litter size on PNDs 1, 4, 7, 11 and 13.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
LOAEC
Effect level:
<= 10 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Key result
Dose descriptor:
NOAEC
Effect level:
>= 20 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
Dose descriptor:
NOAEC
Effect level:
< 10 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
Remarks on result:
other: not determinable due to adverse toxic effects at lowest dose/concentration tested
Dose descriptor:
LOAEC
Effect level:
> 20 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
There were no test item-related litter observations.
Any observations noted were not considered to be treatment-related due to their common occurrence in this age and species of laboratory animal and/or lack of relation to exposure level.

Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There were no test item-related effects on viability indices on PNDs 4, 7, 11 and 13, post implantation survival index or live birth index (%).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were test item-related decreases in pup body weights on PNDs 7 (F1 females), and PND 11 and PND 13 (F1 males and females) and in body weight change for intervals PNDs 4 to 7, 7 to 11, and 11 to 13 (F1 males and females) at all exposure levels. By PND 13, mean test item-exposed pup body weights were 22.5% and 27.2% less than the control pup body weights for males and females, respectively. The test item-related decreases in pup body weights occurred at the same intervals when the dams in the treated groups had decreased body weights, decreased body weight gain and decreased food consumption, therefore the decreased pup body weights were considered secondary to the maternal systemic toxicity.

Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Description (incidence and severity):
Thyroid/Parathyroid - F1 Pups
The thyroid and parathyroid of F1 pups were microscopically normal.
Other effects:
no effects observed
Description (incidence and severity):
Anogenital Distance and Nipple Retention
There were no test item-related effects on ano-genital distance (F1 males and females) or nipple retention (F1 males only).

Sex ratio
There were no test item-related effects on pup sex ratios on PNDs 1, 4, 7, 11 and 13 or body weight on PNDs 1 and 4 (F1 males and females) and 7 (F1 males).

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
>= 20 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: Test item-related organ weight changes (% difference relative to controls) in rats exposed to Nicotine

Group/sex

2M

3M

4M

2F

3F

4F

Exposure (µg/L)

10

15

20

10

15

20

Adrenals

 

 

 

 

 

 

    Absolute weight (%)

+11

+17

+13

+41

+13

+31a

    vs. body weight (%)

+15

+19a

+20a

+49a

+19a

+26a

    vs. brain weight (%)

+11

+20a

+15a

+40a

+14a

+31a

 

 

 

 

 

 

 

Pituitary

 

 

 

 

 

 

    Absolute weight (%)

+6

+18a

+19a

-

-

-

    vs. body weight (%)

+10

+23a

+30a

-

-

-

    vs. brain weight (%)

+6

+20a

+22a

-

-

-

 

 

 

 

 

 

 

Spleen

 

 

 

 

 

 

    Absolute weight (%)

-14

-13

-24a

-11

-26

-14

    vs. body weight (%)

-9

-11

-18a

-7

-22

-16a

    vs. brain weight (%)

-13

-10

-22a

-11

-25

-13

 

 

 

 

 

 

 

Thymus

 

 

 

 

 

 

    Absolute weight (%)

-21

-13

-36

-28

-26

-10

    vs. body weight (%)

-19

-12

-31

-27

-22

-14

    vs. brain weight (%)

-22

-12

-36

-28

-25

-9

 

 

 

 

 

 

 

Prostate

 

 

 

 

 

 

    Absolute weight (%)

-13

-19a

-23a

n/a

n/a

n/a

    vs. body weight (%)

-6

-14

-13

n/a

n/a

n/a

 

 

 

 

 

 

 

Uterus/Cervix

 

 

 

 

 

 

    Absolute weight (%)

n/a

n/a

n/a

-24a

-31a

-28a

    vs. body weight (%)

n/a

n/a

n/a

-19a

-27a

-30a

aStatistically significant difference between mean values for test item-treated and control groups.

-= not test item-related,  n/a not applicable, vs. brain weight not was measured for the prostate, uterus/cervix.

Terminal Necropsy

Liver

Hepatocellular necrosis (minimal to slight) occurred sporadically in the liver of terminal necropsy males at ≥ 15 µg/L and females at 10 and 20 µg/L. Necrosis was associated with minimal to slight neutrophilic to mixed inflammatory cell infiltrates and correlated with clinical chemistry findings of increased alanine aminotransferase activity in males at ≥ 15 µg/L. Marked hydropic degeneration was also present in 1 terminal necropsy female at 20 µg/L. Hepatocellular necrosis and hydropic degeneration were not present in 15 µg/L females surviving to terminal sacrifice, but they were present in 5 of the 15 µg/L unscheduled decedents. Although the findings did not exhibit a clear relationship to exposure level, they were considered to be related (directly or indirectly) to the test item, because they did not occur in the control groups and were present (at up to marked severity) in most of the unscheduled decedents at 15 µg/L.

Test item-related findings in the liver in rats exposed toNicotine

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure (µg/L)

0

10

15

20

0

10

15

20

Necrosis, Hepatocellular

 

 

 

 

 

 

 

 

Minimal

0

0

0

0

0

0

0

1

Slight

0

0

2

1

0

2

0

0

Total

0

0

2

1

0

2

0

1

Degeneration, Hydropic

 

 

 

 

 

 

 

 

Marked

0

0

0

0

0

0

0

1

Infiltrate, Inflammatory Cell

 

 

 

 

 

 

 

 

Minimal

0

0

0

1

0

0

0

1

Slight

0

0

2

1

0

2

0

0

Total

0

0

2

1

0

2

0

1

 

 

 

 

 

 

 

 

 

Number of tissues examined

5

5

5

5

5

5

2

5

 

Secondary findings

Adrenal Gland

Diffuse cortical hypertrophy (minimal to moderate), primarily involving the zona fasciculata, was present in the adrenal gland in both sexes at ≥ 10 µg/L. Changes were generally greater in incidence and severity in females than in males.

Test item-related findings in the adrenal gland in rats exposed toNicotine

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure (µg/L)

0

10

15

20

0

10

15

20

Hypertrophy, Cortical, Diffuse

 

 

 

 

 

 

 

 

Minimal

0

0

2

0

0

0

0

0

Slight

0

3

1

3

0

4

1

2

Moderate

0

0

0

0

0

1

0

2

Total

0

3

3

3

0

5

1

4

 

 

 

 

 

 

 

 

 

Number of tissues examined

5

5

5

5

5

5

2

5

 

Thymus

Thymic atrophy (minimal to marked) occurred in both sexes in all groups, including a female control, but was increased in incidence and/or severity compared to the controls in both sexes at ≥ 10 µg/L. Changes were generally greater in incidence and/or severity in females than in males.

Test item-related findings in the thymus in rats exposed toNicotine

Group/sex

1M

2M

3M

4M

1F

2F

3F

4F

Exposure (µg/L)

0

10

15

20

0

10

15

20

Atrophy

 

 

 

 

 

 

 

 

Minimal

0

4

2

3

0

0

0

1

Slight

0

0

2

1

1

1

0

1

Moderate

0

0

0

0

0

3

0

2

Marked

0

0

0

0

0

1

2

1

Total

0

4

4

4

1

5

2

5

 

 

 

 

 

 

 

 

 

Number of tissues examined

5

5

5

5

5

5

2

5

 

Axillary Lymph Nodes

Plasma cells were present in all groups including the controls but were increased in incidence and severity, compared to the controls, in both sexes at 10 and 20 µg/L but not at 15 µg/L. The finding did not exhibit an exposure relationship and was considered to be unrelated to the test item. 

All other microscopic findings occurred sporadically or at similar incidence and severity in control and test item-exposed groups and were considered incidental and due to biological variability.

Analytical Chemistry

The concentration results for Nicotine dose formulations of all groups analyzed during the course of the study met the study plan specified acceptance criteria. Therefore, all dose formulations used for exposures in this study met the acceptance criteria required by the study plan.

Summary of Dose confirmation results (Mean%Nominal)

 

Group 2

Group 3

Group 4

Interval

(2.6 mg/mL)

(3.3 mg/mL)

(5.2 mg/mL)

 

 

 

 

Week 1

102.1

102.5

103.1

 

 

 

 

 

Group 2

Group 3

Group 4

Interval

(2.6 mg/mL)

(3.5 mg/mL)

(4.8 mg/mL)

 

 

 

 

Week 2

102.8

102.8

101.7

Week 3

102.6

102.6

102.6

 

 

 

 

 

Group 2

Group 3

Group 4

Interval

(2.6 mg/mL)

(3.5 mg/mL)

(5.0 mg/mL)

 

 

 

 

Week 4

102.1

102.7

103.1

Week 5

100.0

99.0

101.1

 

 

 

 

 

Group 2

Group 3

Group 4

Interval

(2.3 mg/mL)

(3.5 mg/mL)

(5.0 mg/mL)

 

 

 

 

Week 6

99.4

100.8

101.7

Week 7

101.2

100.4

99.7

Week 8

103.9

103.2

104.0

Week 9

104.6

101.6

104.9

Week 10

100.9

101.3

102.5

Week 11

-

-

102.1

 

 

 

 

Exposure System Monitoring Results

Mean (S)-Nicotine vapor/aerosol concentrations are summarized below:

Group

Number of Exposures

Vapor/Aerosol Concentration (µg/L)

Target

Analytical

1

63

0 (air only)

0

2

68

10

10.87

3

63

15

14.44

4

73

20

20.29

No Nicotine was detected in Group 1 samples. Mean achieved analytical concentrations for Group 2 was 9% above target, Group 3 was 4% below target and Group 4 was 1% above target.

A single particle sizing sample was collected for Group 4 during animal exposure in Week 1. No test item was detected on stages 3 to 8 with test item detected only in the final stage impinger confirming the results obtained during pre-study trials that the test atmospheres were essentially vapor only. Requirement for particle size distribution measurements were then removed by study plan amendment.

Applicant's summary and conclusion

Conclusions:
Inhalation exposures of Nicotine to rats for up to 5 weeks in males (during premating, mating and post-mating) and for up to 10 weeks in females (during premating, mating, gestation and lactation) at target exposure levels of 10, 15 and 20 µg/L resulted in adverse effects at all exposure levels on adult males and females for clinical signs (including mortality), body weights and food consumption.
Hepatocellular necrosis and/or hydropic degeneration occurred at ≥ 10 ug/L in unscheduled decedents and animals surviving to terminal sacrifice. Although the findings did not exhibit a clear relationship to exposure level, they were considered to be related (directly or indirectly) to the test item, because they did not occur in the control groups and were present (at up to marked severity) in most of the unscheduled decedents at 15 µg/L. Microscopic changes secondary to Nicotine-associated chronic stress occurred at ≥ 10 µg/L in the adrenal gland (cortical hypertrophy), thymus (atrophy) and uterus (atrophy) and correlated with stress-associated organ weight changes. Stress associated microscopic changes were generally greater in incidence and/or severity in females than males and clinical findings resulting in welfare euthanasia occurred only in females. This likely reflects the longer duration of the test item exposure period for females. The pathology findings were not by themselves considered adverse except when viewed in conjunction with the mortality and other adverse effects seen in this study.
There were test item-related effects on parental female estrous cycling at all exposure levels. Specifically, the percentages of irregular cycles ranged from 22 to 40% for the test item-treated females. The mean cycle lengths increased for all test item-treated females from 2.1 to 2.8 x control. Despite the test item-related effect on estrous cycle number and duration, there was no impact on mating and fertility and therefore these effects were considered non-adverse.
There were adverse test item-related decreases in pup body weights on PNDs 7 (F1 females), and 11 and 13 (F1 males and females) and in body weight change for intervals PND 4 to 7, 7 to 11, and 11 to 13 (F1 males and females) at all exposure levels. By PND 13 at 20 µg/L, mean test item-exposed pup body weights were 22.6% and 27.2% less than the control pup body weights for males and females, respectively. The test item-related decreases in pup body weights occurred at the same intervals when the dams in the treated groups had decreased body weights, decreased body weight gain and decreased food consumption, therefore the decreased pup body weights were considered secondary to the maternal systemic toxicity.
The male and female parental systemic No Observed Adverse Effect Level (NOAELsystemic, F0) was not considered to have been determined because adverse effects were seen at all exposure levels in the parental animals, thus a Lowest Observed Adverse Effect Level (LOAELsystemic, F0) for subacute repeated dose administration was determined to be 10 µg/L. A parental reproductive No Observed Adverse Effect Level (NOAELreproductive, F0) was determined to be ≥20 µg/L Nicotine based on no adverse effects on estrous cyclicity, Mating and Fertility parameters, the number of implantation sites, gestation length, total litter size, and live litter size. This determination was also based on the determination that the test item-related effect on estrous cycle number and duration were considered non-adverse.
The F1 No Observed Adverse Effect Level on growth and developmental toxicity (NOAELdevelopmental, F1) was determined to be ≥20 µg/L Nicotine based on no adverse effects on mortality, litter observations, pup sex ratios, ano-genital distance (F1 males and females) or nipple retention (F1 males only), neurobehavioral evaluations (Functional Observational Battery and Motor Activity), and endocrine (Thyroid Hormone) assay. This determination was also based on the decreased pup body weights recorded which were considered secondary to the maternal systemic toxicity.
Executive summary:

Sprague-Dawley CD®rats (10 animals/sex/group) were exposed by nose-only inhalation once daily to 0 (air control), 10, 15 or 20 µg/L of (S)-Nicotinevapor/aerosolfor 6 hours per day. The Fmales were exposed during the pre-cohabitation, cohabitation and post-mating periods (approximately 35 days) and then euthanized and necropsied. The F0females were exposed during the pre-cohabitation and cohabitation periods and during gestation (Gestation Day [GD] 0 through GD 19) and lactation (Lactation Day [LD] 5 through LD 13) for approximately 70 days and then euthanized and necropsied. Parameters evaluated during the study for the F0animals were: viability, clinical observations, body weights, food consumption, estrous cycling, mating and fertility, parturition and littering, functional assessments, clinical pathology (termination), hormone analysis, organ weights, macroscopic observations and microscopic pathology. Parameters evaluated for the F1 pups were: viability, clinical observations, sex, body weights, ano-genital distance, nipple retention, hormone analysis, macroscopic observations and microscopic examination of the thyroid gland.

Mean Nicotine vapor/aerosol concentrations are summarized below:

Group

Number of Exposures

Vapor/Aerosol Concentration (µg/L)

Target

Analytical

1

63

0 (air only)

0

2

68

10

10.87

3

63

15

14.44

4

73

20

20.29

The test atmospheres were essentially vapor only.

Inhalation exposures of Nicotine to rats for up to 5 weeks in males (during premating, mating and post-mating) and for up to 10 weeks in females (during premating, mating, gestation and lactation) at target exposure levels of 10, 15 and 20 µg/L resulted in adverse effects at all exposure levels on adult males and females for clinical signs (including mortality), body weights and food consumption.

Hepatocellular necrosis and/or hydropic degeneration occurred at ≥ 10 ug/L in unscheduled decedents and animals surviving to terminal sacrifice. Although the findings did not exhibit a clear relationship to exposure level, they were considered to be related (directly or indirectly) to the test item, because they did not occur in the control groups and were present (at up to marked severity) in most of the unscheduled decedents at 15 µg/L. Microscopic changes secondary to Nicotine-associated chronic stress occurred at ≥ 10 µg/L in the adrenal gland (cortical hypertrophy), thymus (atrophy) and uterus (atrophy) and correlated with stress-associated organ weight changes. Stress associated microscopic changes were generally greater in incidence and/or severity in females than males and clinical findings resulting in welfare euthanasia occurred only in females. This likely reflects the longer duration of the test item exposure period for females. The pathology findings were not by themselves considered adverse except when viewed in conjunction with the mortality and other adverse effects seen in this study.

There were test item-related effects on parental femaleestrous cycling at all exposure levels. Specifically, the percentages of irregular cycles ranged from 22 to 40% for the test item-treated females. The mean cycle lengths increased for all test item-treated females from 2.1 to 2.8 x control. Despite the test item-related effect on estrous cycle number and duration, there was no impact on mating and fertility and therefore these effects were considered non-adverse.

There were adverse test item-related decreases in pup body weights on PNDs 7 (F1females), and 11 and 13 (F1males and females) and in body weight change for intervals PND 4 to 7, 7 to 11, and 11 to 13 (F1males and females) at all exposure levels. By PND 13 at 20 µg/L, mean test item-exposed pup body weights were 22.6% and 27.2% less than the control pup body weights for males and females, respectively. The test item-related decreases in pup body weights occurred at the same intervals when the dams in the treated groups had decreased body weights, decreased body weight gain and decreased food consumption, therefore the decreased pup body weights were considered secondary to the maternal systemic toxicity. 

The male and female parental systemic No Observed Adverse Effect Level (NOAELsystemic, F0) was not considered to have been determined because adverse effects were seen at all exposure levels in the parental animals, thus a Lowest Observed Adverse Effect Level (LOAELsystemic, F0) for subacute repeated dose administration was determined to be 10 µg/L.  A parental reproductive No Observed Adverse Effect Level (NOAELreproductive, F0) was determined to be ≥20 µg/L (S)-Nicotine based on no adverse effects on estrous cyclicity, Mating and Fertility parameters,the number of implantation sites, gestation length, total litter size, and live litter size. This determination was also based on the determination that the test item-related effect on estrous cycle number and duration were considered non-adverse.

The F1 No Observed Adverse Effect Level on growth and developmental toxicity (NOAELdevelopmental, F1) was determined to be ≥20 µg/L Nicotine based on no adverse effects on mortality, litter observations, pup sex ratios, ano-genital distance (F1males and females) or nipple retention (F1males only), neurobehavioral evaluations (Functional Observational Battery and Motor Activity), and endocrine (Thyroid Hormone) assay. This determination was also based on the decreased pup body weights recorded which were considered secondary to the maternal systemic toxicity.