Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Aug 2019 - 04 Sep 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
adopted June 2018
Deviations:
no
GLP compliance:
yes
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS:

The study design was based on a final ECHA decision (TPE-D-2114394286-38-01/F).

- Premating exposure duration for parental (P0) animals : Ten weeks premating exposure duration was required because there is no substance specific information in the dossier supporting shorter premating exposure duration as advised in the ECHA Guidance on information requirements and chemical safety assessment ECHA Chapter R.7a, Section R.7.6 (version 6.0, July 2017).

- Basis for dose level selection: The highest dose level was chosen to induce some toxicity to allow comparison of effect levels and effects of reproductive toxicity with those of systemic toxicity. The dose level selection should be based upon the fertility effects with the other cohorts being tested at the same dose levels. As there was no relevant data to be used for dose level setting available, a range-finding study was performed.

- Inclusion/exclusion of extension of Cohort 1B: ECHA concluded that Cohort 1B shall not be extended to include mating of the animals and production of the F2 (for details see ECHA decision).

- Termination time for F2 : a F2 generation was not requested by ECHA and accordingly not included in the study

- Inclusion of developmental neurotoxicity Cohorts 2A and 2B : Based on the available data, ECHA considered that the criteria to include Cohorts 2A and 2B were not met and concluded that the developmental neurotoxicity Cohorts 2A and 2B need not to be included.

- Inclusion of developmental immunotoxicity Cohort 3 : Based on the available data, ECHA considered that the criteria to include Cohort 3 were not met and concluded that the developmental immunotoxicity Cohort 3 need not to be included.

- Route of administration : inhalation

- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: The study was performed in the rat, as this is the default species according to the OECD guideline 443

Test material

Constituent 1
Chemical structure
Reference substance name:
Chloroethane
EC Number:
200-830-5
EC Name:
Chloroethane
Cas Number:
75-00-3
Molecular formula:
C2H5Cl
IUPAC Name:
chloroethane
Test material form:
gas

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD (SD)
Details on species / strain selection:
The Sprague Dawley 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, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: P animals 6-9 wks; F1 animals 3 wks
- Weight at study initiation: (P) Males: 293-385 g; Females: 133-196 g; (F1) Males: 35.1-60.8 g; Females: 34.9-61.4 g
- Housing: P animals were initially housed 2 or 3 per cage by sex (unless numbers were reduced by mortality) in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms. A few days prior to mating, P males were transferred to individual cages with solid bottoms. Females were transferred to these cages for mating. Mated females were transferred to individual solid bottomed cages and white paper tissue was also supplied as nesting material from Gestation Day (GD) 20. F0 females with their litters were retained in this type of cage until termination. On a suitable day after the completion of mating, the F0 males were re-housed with their original cage mates. For GD 22 to 24 blood sampling, F0 dams were fasted separately from their unselected pups which were housed in appropriately sized cages. F1 animals retained after weaning were housed 2 or 3 per cage by sex in appropriately sized suspended polycarbonate cages with stainless steel grid tops and solid bottoms.
- Diet: SDS VRF-1 breeder diet (Special Diet Services, Essex, UK), ad libitum
- Water: water from the public supply in water bottles, ad libitum
- Acclimation period: at least 2 weeks before commencement of dosing
- Conditioning: for at least 4 days before commencement of dosing, all P and F1 animals were conditioned to the restraint procedure used for snout only exposure by placing the animals in the restraint tubes for gradually increasing periods of time, up to the maximum expected duration of 6 hours daily used on this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 25
- Humidity (%): 19 - 89
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 Aug 2019 To: 28 Feb 2020

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Details on exposure:
ATMOSPHERE GENERATION:
Test atmosphere was generated using compressed gas (chloroethane) diluted with dry compressed air. To maintain a consistent flow and head of gas in the cylinder, heating jackets were used to heat the cylinder and the metal pipework to a temperature between 20 and 40°C. This temperature was monitored and recorded at regular intervals throughout each exposure period.

Inhalation exposures were conducted in a room adjacent to, but separate from, the animal holding room. Exposures to the test atmospheres were performed using appropriately sized modular snout only stainless steel flow past systems (in-house design). This exposure technique allowed a continuous supply of test atmosphere to be delivered to each animal; the biased flow ensured that there was no re-breathing of the test atmosphere. Separate inhalation exposure systems were used for the delivery of control and test atmospheres. Each inhalation exposure system was located in an extract booth (for the protection of personnel and to prevent cross-group contamination) and was operated to sustain a dynamic air flow sufficient to ensure an evenly distributed exposure atmosphere.
All air flow rates (delivered and extracted) were controlled and monitored visually using calibrated flow meters. Chamber air flow rates, temperature and relative humidity levels were monitored and recorded at regular intervals during each daily exposure period.
For inhalation exposure, the animals were restrained in clear, tapered, polycarbonate tubes with an adjustable back-stop to prevent the animals from turning in the tubes. The animals’ snouts protruded through the anterior end of the restraint tubes which were connected to the exposure chamber by way of a push-fit through an inhalation port in the chamber wall. This exposure technique was used to minimise concurrent exposure by the oral and dermal routes.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: max.14 nights
- Proof of pregnancy: vaginal plug and/or sperm in vaginal lavage referred to as day 0 of pregnancy
- Further matings after one unsuccessful attempt: no
- After successful mating each pregnant female was caged individually in solid bottomed cages and white paper tissue (supplied as nesting material from Gestation Day (GD) 20
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test and control atmospheres were sampled using Tedlar(R) gas sampling bags with PTFE connections within a vac-U chamber box connected to a vacuum pump. No gravimetric analysis was undertaken and the volume of gas sampled was not recorded. On study days 1-7, all groups were sampled daily; from week 2, group 1 was sampled weekly and groups 2-4 were sampled daily. On study day 21 group 3 was samples once. After sampling, retained samples were stored at ambient temperature. Chemical analysis was undertaken on all retained Group 2-4 samples to determine the atmosphere concentration of the test item. One sample taken for Group 1 was analysed and a further sample was retained for possible analysis and discarded once absence of the test item in the first sample was confirmed.
The retained atmosphere samples were subjected to chemical analysis using validated GC-FID methods.

P animals
The overall low dose mean atmosphere concentration (2500 ppm) was within target specification. Variability was slightly above the upper specification limit of 15% (16.8% RSD). The overall Group 3 mean atmosphere concentration (17200 ppm) on Days 1 and 2 was 145% above the target of 7000 ppm due to an error in instruction. Over Days 3-6, with Group 3 now designated as the high dose (originally intended as the intermediate dose), mean atmosphere concentration (14100 ppm) was within target specification and this was achieved with low variability (3.3% RSD). This phase of dosing was intended to acclimatise the animals to high atmosphere concentrations of chloroethane before proceeding to 20000 ppm target atmosphere concentration. Over Days 7-18 mean atmospheric concentration (17100 ppm) was below target specification and was achieved with high variability (26.9% RSD). From Day 23 onwards, the Group 3 target atmosphere concentration was reduced from 20000 ppm back to 15000 ppm due to clinical signs observed. The Group 3 mean atmosphere concentration (14900 ppm) was within target specification and achieved with acceptable variability (12.2% RSD).
The overall Group 4 mean atmosphere concentrations (20800 ppm) was above target specification on Days 1 and 2 due to an error in instruction. Group 4 animals were then placed on a dosing holiday on Days 3-6. On re-commencement of dosing (Day 7 onwards), Group 4 was assigned as the intermediate dose group (originally intended as the high dose group). From Day 7 onwards, the Group 4 mean atmosphere concentration (7040 ppm) was within target specification and this was achieved with low variability (11.2% RSD). A summary of achieved concentrations is presented in Table 4 under 'Any other information on results incl. tables'.

F1 animals
The Group 2 mean achieved atmosphere concentration was within target specification (2480 ppm), this was achieved with low variability (13.9% RSD). The Group 3 mean achieved atmosphere concentrations up to Day 18 of dosing of F0 animals was the same as the overall achieved mean achieved atmosphere concentrations. From Day 23 onwards of dosing the mean atmosphere concentration was within target specification (15200 ppm), this was achieved with low variability (13.4% RSD). The Group 4 mean atmosphere concentration on Days 1 and 2 of dosing of F0 animals was the same as the overall achieved mean atmosphere concentration. From Day 7 onwards of dosing the mean achieved atmosphere concentration was within target specification (6870 ppm), this was achieved with low variability (12.8% RSD). A summary of achieved concentrations is presented in Table 5 under 'Any other information on results incl. tables'.

A summary of the overall achieved concentrations during the study (P and F1 animals) is presented in Table 6 under 'Any other information on results incl. tables'.
Duration of treatment / exposure:
P Males: At least 10 weeks prior to mating and then up to necropsy (total of 18 weeks)
P Females: At least 10 weeks prior to mating, then through mating, gestation and at least until LD 21. (P Groups 1 and 2 up to Day 71, P Groups 3 and 4 up to Day 75).
Cohort 1A and 1B: PND 21 up to at least PND 84 or PND 98, respectively.

A few animals were not dosed on individual days due to animal welfare reasons. These dosing holidays were considered to have had no effect on the study outcome as the number of dosing days missed per animal was a very small proportion of the scheduled dosing period and was necessary for animal welfare and veterinary treatment (for details see Table 7 under 'Any other information on results incl. tables').

The P females were not dosed on GD 20/21 until after their litters were born and then exposure duration was initially reduced to allow the dams to acclimatise to being away from their litter. The F0 females were then dosed as follows:
LD 1 to 2: approximately 1 hour per day
LD 3 to 4: approximately 4 hours per day
LD 5 until prior to termination: approximately 6 hours per day
Frequency of treatment:
6 h/day, 7 days/week
Details on study schedule:
- F1 animals were not mated in this study.
- Age at mating of the mated animals in the study: P animals were approximately 16 and 19 weeks of age at start of mating.
Doses / concentrationsopen allclose all
Dose / conc.:
2 500 ppm
Remarks:
corresponding to 6692 mg/m³
Dose / conc.:
7 000 ppm
Remarks:
corresponding to 18739 mg/m³
Dose / conc.:
15 000 ppm
Remarks:
corresponding to 40154 mg/m³
No. of animals per sex per dose:
20 males and 20 females (for P as well as F1 animals)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The inhalation route of administration was selected for this study as the test item is a gas and this is the only route of possible human exposure.
In the previous dose range finding study, male rats were dosed for at least 60 days and female rats were dosed for 2 weeks prior to mating, throughout mating and gestation until at least LD 21 at concentration levels of 0, 5000, 10000 or 20000 ppm. At 20000 ppm, one female was found dead on Day 2, during dosing. Furthermore, on Day 2 of dosing, animals dosed at 20000 ppm had reduced or no reaction to touch, pale tails and were cold to touch. Up to Day 3 of dosing, rectal body temperatures at 20000 ppm were lower than controls directly following exposure. In all treatment groups, the males had lower food consumption and F1 pups had a lower body weight from LD 7 to 21, when compared to the controls. No effects were seen on any fertility and reproduction parameters or on any litter data/survival rates at any concentration.
Based on the dose range finding study results, a high concentration level of 20000 ppm, but with a reduced level of 15000 ppm for Days 1 to 3 of dosing, was considered to be a suitable dosing regimen. A reduced level of 15000 ppm was to allow the high concentration level animals to acclimatise to the test item over the first few days of dosing. The low and intermediate concentration levels of 2500 ppm and 7000 ppm were selected.

On Day 1 of dosing, abnormal behaviour of agitation, licking and/or chewing was observed in six P Group 4 females (target concentration level 15000 ppm), resulting in self-inflicted injuries of five females to their forepaws. These animals were removed from inhalation dosing as soon as the injuries were noted (approximately 4 hours into the 6 hour inhalation period) and dosing on Day 1 was stopped for all Group 4 animals. On Days 1 and 2, these behaviours were also noted for some other animals in Groups 3 and 4 which were removed from dosing as soon as the behaviour was noted and before injury could occur, and subsequently, all Group 4 animals were again removed from dosing on Day 2. Analytical results of test atmospheres received on Day 2 showed over dosing of the Group 4 animals (up to 21900 ppm) and they were given a dosing holiday on Days 3 to 6 before dosing recommenced on Day 7. It was found that on Days 1 and 2, the inhalation settings had been incorrect for all test item groups resulting in the atmosphere concentrations being greater than those targeted for Groups 3 (up to 18000 ppm) and 4 (over 21900 ppm). The five self-injured Group 4 females were not dosed after Day 1 and were sent for unscheduled necropsy and replaced by five spare females.

After discussions with the Sponsor, Home Office Project Licence Holder and Home Office Named Veterinarian, the concentration levels for Group 3 and 4 animals were changed from Day 3 i.e. Group 3 became the high dose group as their achieved atmosphere concentrations on Days 1 and 2 were close to that initially targeted for the high concentration level (15000 ppm) and Group 4 were given a 4 day dosing holiday and started dosing again on Day 7 at the intermediate concentration level of 7000 ppm.

On Day 18, abnormal behaviour of agitation, licking and/or chewing was seen in five F0 Group 3 animals (target concentration level 20000 ppm) after approximately 3 hours dosing, resulting in self-inflicted injuries to the forepaws and these animals were sent for unscheduled necropsy. Consequently, all Group 3 animals were removed from dosing on Day 18. Analytical results of test atmospheres for Group 3 on Day 18 showed a low concentration when the first atmosphere sample was taken and the second sample showed the concentration to be on target for 20000 ppm. It was found that for the initial hour in the dosing period, the gas cylinder valves were not turned on correctly as confirmed by the low atmosphere concentration; the second atmosphere sample was taken approximately 40 minutes after the valve settings were corrected. After discussion with the Sponsor Monitor, Group 3 animals were not dosed on Days 19 to 22 and the concentration level for Group 3 was reduced to 15000 ppm from Day 23, as the concentration of 20000 ppm using snout only inhalation appeared to be close to the concentration that induced stereotypical behaviour i.e. agitation, licking and/or chewing.
These events affected the P animals in Groups 3 and 4 over the short term and because after a 4 day dosing holiday they were considered suitable to continue with dosing, it was considered that there was no adverse impact on the study outcome. The animals in Groups 3 and 4 were dosed up to Day 75 during the prepairing period so that they were given a total of 10 weeks dosing.

- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical biochemistry: animals were fasted prior to blood sampling; those P females that were sent for unscheduled necropsy because they had not littered by GD 24, were not fasted prior to blood sampling as they were potentially pregnant
Positive control:
not applicable

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Mortality/Moribundity Checks: twice daily, once at the start and once towards the end of the working day throughout the study for general health/mortality and moribundity
- Pre- and post-dose observations: prior to dosing and regularly throughout the day on each day of dosing for signs of reaction to treatment, with particular attention being paid to the animals during and for the first hour after dosing; from Day 46, post-dose observations for F0 generation were limited to immediately post-dose unless further observations were required

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least weekly, beginning Week -1

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed weekly beginning Week -1; females were weighed weekly beginning Week -1 until pairing for mating and then on GD 0, 7, 14 and 20 and on Lactation day (LD) 1, 4, 7, 14 and 21.

FOOD CONSUMPTION: Yes
- Food consumption for each cage/animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: on a regular basis throughout the study by visual inspection of the water bottles

HAEMATOLOGY AND COAGULATION
- Blood was collected via the jugular vein from P and F1A animals (10 rats/sex/group) in the morning of scheduled sacrifice. Animals were fasted prior to blood sampling. The following parameters were analysed: 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).

The following coagulation parameters were detrmined: activated partial thromboplastin time, fibrinogen, prothrombin time, sample quality.

CLINICAL CHEMISTRY
- Blood was collected via the jugular vein from P and F1A animals (10 rats/sex/group) in the morning of scheduled sacrifice. Animals were fasted prior to blood sampling. The following parameters were analysed: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, gamma-glutamyltransferase, creatine kinase, total bilirubin (when total bilirubin was >8.55 µmol/L, indirect and direct bilirubin were also measured), urea, creatinine, calcium, phosphate, total protein, albumin, globulin, albumin/globulin ratio, glucose, cholesterol, triglycerides, sodium, potassium, chloride, sample quality.

THYROID HORMONES
- Blood was collected via the jugular vein from P and F1A animals (10 rats/sex/group) in the morning of scheduled sacrifice. Animals were fasted prior to blood sampling. Blood samples were processed to serum. T3 and T4 were determined on Advia Centaur CP Immunoassay System by using solid phase, competitive chemiluminescent enzyme immunoassays. TSH was measured by using a solid phase enzyme immunometric assay – ELISA kit manufactured by BioVendor, Cat. No. RTC007R.

URINALYSIS
- Urine was sampled over 6 h with absence of food and water from P and F1A animals (10 rats/sex/group) during the last week of dosing. The following parameters were analysed: colour, appearance/clarity, specific gravity, volume, pH, protein, glucose, bilirubin, ketones, blood.

RECTAL TEMPERATURES
- Rectal temperatures were taken as soon as possible after completion of dosing on Days 1 to 2 from P generation Group 4 animals to ascertain if there were any changes in body temperature, due to the nature of the test item. Rectal temperatures were taken as soon as possible after the completion of dosing on Day 1 from P generation Group 1 animals, for comparison purposes.
Oestrous cyclicity (parental animals):
Vaginal lavages were taken early each morning and the stages of oestrus observed were recorded from Day 57 (Groups 1 and 2) or Day 61 (Groups 3 and 4) of dosing 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 estrous cycle to allow correlation with histopathology of the ovaries.
Sperm parameters (parental animals):
Parameters examined in P males:
testis weight, epididymis weight, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility (percentage motile sperm, percentage of progressively motile sperm and the straight-line velocity), sperm morphology (control, mid and high dose males only)
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); from excess pups blood samples for thyroid hormones were taken before they were killed and macroscopically examned (externally and internally).

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, sexual maturation (age and body weight at vaginal opening, age and body weight at balano-preputial separation. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development; gross evaluation of external genitalia

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead when possible

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: not performed

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY:
Spleen samples (approximately half of the spleen) were taken from 10 rats per sex per group from the F1 Cohort 1A adults at necropsy and were analysed using a validated analytical method for the relative percentage of T cells, B cells and Natural Killer cells.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals Days 123-130
- Maternal animals: All surviving animals between LD 22 and 24

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

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues indicated in Tables 1 and 2 (under 'Any other information on material and methods incl. tables' were prepared for microscopic examination and weighed, respectively.
For each ovary, 6 step serial sections were taken. One section was stained with haematoxylin and eosin and 5 sections were stained for Immunohistochemistry using PCNA marker for enumeration of primordial and primary follicles The examination of the ovaries included quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.

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 test item-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.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as F1A or F1B animals were sacrificed at 22 to 24 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
From each litter, 1 male and 1 female pup (where they were available) were necropsied from up to 10 litters/group. This consisted of external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of selected tissue (brain, mammary gland, thyroid gland, liver, spleen, thymus and gross lesions) and any grossly abnormal tissues were preserved in 10% formalin or another appropriate fixative. Only mammary glands were histopathologically examined.
The remaining pups in each litter were checked for externally visible abnormalities at the time of killing. Any found to have an abnormality had a gross necropsy performed and any abnormalities were preserved in 10% formalin or another appropriate fixative. The remaining carcasses were then discarded.

At sacrifice of F1A and F1B animals a complete gross necropsy was performed, 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.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
- The tissues indicated in Tables 1, 2 and 3 under 'Any other information on materials and methods incl. tables' were prepared for microscopic examination and weighed, respectively.

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 test item-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.

IMMUNOPHENOTYPING
- Spleen samples (approximately half of the spleen) were taken from 10 rats per sex per group from the F1A adults at necropsy and were analysed for immune cell populations using a validated analytical method.

SPERM ANALYSIS
- Parameters examined in F1A cohort males: testis weight, epididymis weight, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility (percentage motile sperm, percentage of progressively motile sperm and the straight-line velocity), sperm morphology (control, mid and high dose males only)
Statistics:
All results presented in the tables of the report are calculated using non-rounded values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.
All statistical analyses have been performed within the respective study phase, unless otherwise noted. Numerical data collected on scheduled occasions have been summarised and statistically analysed as indicated below according to sex and occasion or by litter. 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, unless otherwise noted.
The pairwise comparisons of interest were as follows: Group 2 vs. Group 1, Group 4 vs. Group 1, Group 3 vs. Group 1.
Levene’s test was used to assess the homogeneity of group variances. 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, respectively.
Datasets with 2 groups (the designated control group and 1 other group) were compared using a Dunnett’s test (equivalent to t-test in Nevis 2012 tables) if Levene’s test was not significant or Dunn’s test (equivalent to Wilcoxon Rank-Sum test in Nevis 2012 tables) if it was significant.
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 is calculated from GD 0 to the day the first pup is observed.

Gestation Index = Number of Animals with Live Offspring x 100 / Number of Animals Pregnant

Live Birth Index = Number of Live Newborn Pups x 100 / Number of Newborn Pups

Sex Ratio (% males) = Number of Live Male Pups x 100 / Total Number of Live Pups

Post-Implantation Loss/Litter = (Number of Implants – Total Newborn Pups) x 100 / Number of Implants
Offspring viability indices:
Viability Index = Number of Live Pups on Day 4 Postpartum x 100 / Number of Live Newborn Pups
Lactation Index = Number of Live Pups on Day 21 Postpartum x 100 / Number of Live Pups on Day 4 (post-culling) Postpartum

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During dosing on Day 1 or 2, three mid dose and one high dose female had what were considered to be self-inflicted injuries to their forepaws but the injuries were slight (scabbed and swollen forepaws). The animals were examined, veterinary treatment given and they were considered fit to recommence dosing.

Other clinical observations for animals (listed in Table 7 under 'Any other information on results incl. tables') and which resulted in dosing holidays for those animals were considered to be incidental to the administration of the test item. The clinical observations of fur staining and/or wet fur were considered to be related to the administration procedure.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
P Generation Animals Killed Due to Stereotypical Behaviour
Group 4 on Day 1
Stereotypical behaviour of agitation, licking and/or chewing was observed in five Group 4 animals (target concentration level 15000 ppm) resulting in what were considered to be self-inflicted injuries to their forepaws. These animals were removed from inhalation dosing as soon as the injuries were noted (approximately 4 hours into the 6 hour inhalation period). They were not dosed after Day 1, received veterinary treatment and were replaced on Day 7 and then sent for unscheduled necropsy on Day 17 (an earlier necropsy was not possible due to capacity restrictions), as it was considered that the welfare end-point had been reached. Observations recorded for some of these animals during this period included scabbed and swollen forepaws, digits missing, abnormal gait or limited usage of forepaw. The tissues for these animals were not examined microscopically as they were dosed for a limited period on Day 1 only and were replaced by spare animals.
Investigations concluded that high atmosphere concentrations of chloroethane above 20000 ppm using snout only inhalation appeared to be close to the concentration that induced stereotypical behaviour and therefore these adverse findings were considered to be test item-related.

Group 3 on Day 18
Stereotypical behaviour of agitation, licking and/or chewing was observed in five Group 3 animals (target concentration level 20000 ppm) resulting in what were considered to be self-inflicted injuries to their forepaws. These animals were removed from inhalation dosing as soon as the injuries were noted (approximately 3 hours into the 6 hour inhalation period). They received veterinary treatment but were sent for unscheduled necropsy on Day 18 as it was considered that the welfare end-point had been reached. Observations recorded for some of these animals included increased activity/hypersensitivity, skin lesions and swollen forepaws, digits missing or abnormal gait.
Grossly, these animals had skin scabs on their digits which correlated microscopically with minimal to mild epidermal necrosis (sometimes extending into the dermis); minimal epidermal hydropic degeneration was also often present in the adjacent intact epidermis. This was accompanied by minimal to mild, acute inflammation in the underlying subcutaneous tissue. Bilateral renal pelvic dilation noted for one male, a common incidental finding in rats, was not appreciable microscopically. The three males were also reported to have dark, gritty content in the gastrointestinal tract at necropsy. Investigations concluded that high atmosphere concentrations of chloroethane above 20000 ppm using snout only inhalation appeared to be close to the concentration that induced stereotypical behaviour and therefore these adverse findings were considered to be test item-related.

Animals Examined after Day 18 of Dosing
P Generation
One mid dose female (7000 ppm) died during parturition on GD 22. There were no abnormal clinical observations and no adverse effects on its body weight or food consumption during the gestation period. Gross pathology findings were considered to be agonal changes only. Microscopic evaluation of the intestinal tract was not possible due to the degree of post-mortem change and the cause of death was not determined. However, deaths during parturition are occasionally seen in rats and as this was not in the high concentration level it was considered not to be test item-related.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For P males given 15000 ppm, there was a consistent, slightly lower body weight gain throughout the dosing period, compared with controls, resulting in a statistically significant difference in weight gain of -14% (p≤0.05) on Day 120. There was no effect in males given 2500 or 7000 ppm or for females pre-mating on Day 71 at up to 15000 ppm.
During gestation, females had a lower body weight gain of -9% or -41% (p≤0.01) up to GD 20, compared to controls, at 7000 or 15000 ppm, respectively. There was no effect on female body weight gain at 2500 ppm.
After parturition on LD 1, the mean body weight for females was similar to controls for all test item groups, however, female weight gain was lower than controls up to LD 14. Body weight gain from LD 1 to 21 was lower than controls by -54%, -51% and -97% (p≤0.01) at 2500, 7000 and 15000 ppm, respectively. For details see Attachment "Summary of P animal body weights and body weight gains" under Attached Background Material.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
For males given 15000 ppm, there was a consistent, slightly lower food consumption throughout the dosing period of up to -9%, compared to controls, with some values being statistically significant at p≤0.05. For males given 7000 ppm a slightly higher food consumprion was noted with some values being statistically significant. This higher food consumption was considered incidental due to the lack of a dose dependency. There was no effect in males given 2500 ppm, or for females pre-mating up to Day 71 at concentrations up to 15000 ppm.
During gestation, food consumption for females was slightly lower than controls by up to -13% (p≤0.05) on GD 20 at 15000 ppm. There was no test item-related effect at chloroethane concentrations up to 7000 ppm. During lactation, food consumption was slightly lower than controls for all test item groups, but the difference from controls was greatest at 15000 ppm (p≤0.01) at up to -21%.
For details see Attachment "Summary of P animal food consumption" under Attached Background Material).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No intergroup differences were noted by visual inspection of water bottles.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related haematology or coagulation changes were noted at any chloroethane concentration level administered in samples taken on the morning of necropsy for the P generation animals. All differences in haematology or coagulation parameters were considered not to be test item-related based on their small magnitude, inconsistent direction, absence of a dose response, general overlap of individual values with the range of control and/or were of a magnitude of variation commonly observed in rats.
For details see Attachment "Summary of P animal haematology and coagulation data" under Attached Background Material.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related clinical chemistry changes were noted at any chloroethane concentration level in samples taken on the morning of necropsy for the F0 generation animals.
All differences in clinical chemistry parameters were considered not to be test item-related based on their small magnitude, inconsistent direction, absence of a dose response, general overlap of individual values with the range of control and/or were of a magnitude of variation commonly observed in rats.
For details see Attachment "Summary of P animal clinical biochemistry data" under Attached Background Material.

THYROID HORMONES
There were no test item-related changes noted at up to 15000 ppm chloroethane for samples taken on the morning of necropsy for the F0 generation.
All differences in these parameters were considered not to be test item-related based on their small magnitude, inconsistent direction, absence of a dose response, general overlap of individual values with the range of control and/or were of a magnitude of variation commonly observed in rats. For details see Attachment "Summary of P animal thyroid hormones" under Attached Background Material.

Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on any urine parameter collected during the last week of dosing at up to 15000 ppm for the P or F1 Cohort 1A animals.
All differences in urine parameters were considered not to be test item-related based on their small magnitude, inconsistent direction, absence of a dose response, general overlap of individual values with the range of control and/or were of a magnitude of variation commonly observed in rats.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related microscopic findings. There were, however, microscopic findings of note in several animals which were considered not to be test item-related.
The grossly small testes in one mid dose and two high dose males correlated microscopically with severe diffuse tubular atrophy in one mid and one high dose male. This could not be confirmed in the second high dose male as the right testis was grossly small and only the left testis (in which there was no visible lesion) was available for microscopic examination (as per protocol). The small epididymides in these three males correlated microscopically with severely decreased sperm cellularity, reflecting the histological changes seen in the testes. The prostatic masses noted grossly in one low dose, two mid dose and one high dose male correlated microscopically with mild to moderate, acute to chronic-active inflammation; there was no histological evidence of neoplasia. There was no histological correlate for the small prostate in one mid dose male. Such lesions in the testis, epididymis and prostate gland are recognised as spontaneous background findings in rats and, given the small number of animals affected, were considered not to be test item-related, occurring in a few treated animals only by chance.
Mild follicular cell hyperplasia, as seen in the thyroid gland of one high dose female, can also occur as a spontaneous background finding and as such, was considered not to be test item-related.
All remaining microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be related to the administration of chloroethane.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A malignant astrocytoma was present in the brain of one female receiving 15000 ppm. This tumour may be a spontaneous background finding in Sprague Dawley rats, and given that only a single animal was affected it was considered not to be test item-related.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
BODY TEMPERATURE: Animals in Group 4 were initially administered 15000 ppm and after dosing on Days 1 and 2, males’ rectal temperature was lower by up to 0.8°C, compared with control values on Day 1. On Day 2 only, females’ rectal temperature was lower by 0.7°C, compared with control values on Day 1.
No further measurement of rectal temperatures were made.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no effect on the P generation estrous cycles during the 2 weeks before mating or during the mating period at up to 15000 ppm, compared with controls.
The higher number of P females with major cycle changes at 7000 ppm was considered not to be test item-related as the difference from controls was not concentration level-related - animals given 15000 ppm had similar pattern values to controls.
For details see Attachment "Summary of P females estrous cycle patterns" under Attached Background Material.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
During computer-assisited sperm analysis, the percentage of progressively motile sperm was 29% lower than controls at 15000 ppm with a greater number of males (8/24) having progressive motility of less than 10% (controls 3/25). There were no notable test item-related effects on the percentage of motile sperm or straight line velocity at up to 15000 ppm.
At 15000 ppm, the total number of sperm was slightly lower than control by -18%. Values at 2500 and 7000 ppm were comparable with the control mean. There were no notable effects on sperm morphology at the concentrations examined of 7000 and 15000 ppm. There was no test item-related effect on the number of spermatids.
For details see Attachment "Summary of P male sperm measures" under Attached Background Material.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
MATING PERFORMANCE, FERTILITY AND DURATION OF GESTATION
At 15000 ppm, male and female fertility indices were slightly lower at 86%, compared to a control value of 92%. However, the number of non-siring males and non-pregnant females were similar to controls, as 3 males were not paired with a female as there were insufficient females available. Therefore, there were considered to be no effects on mating performance in the F0 generation at up to 15000 ppm.

The mean duration of gestation for all groups was 22 days, however, there was a slight shift in the individual duration of gestation at 15000 ppm with no litters being born on GD 21 (3 were born for the control group), the majority born on GD 22, a slightly higher number than controls on GD 23 (3 compared to one for controls) and one litter containing 2 pups only on GD 24 (compared to none for controls). There was no effect on the duration of gestation at 2500 or 7000 ppm.

Animals which failed to produce a litter by their expected GD 24 were sent for unscheduled necropsy as listed below. All were not pregnant except for two mid dose femles which each had one very large dead fetus (failure to give birth to a single, large fetus is occasionally seen in rats).

For details see Attachment "Summary of P female mating performance_fertility_duration of gestation_litter performance" under Attached Background Material.

PRE- AND POST-IMPLANTATION LOSS
The number of corpora lutea was unaffected by administration of chloroethane at 15000 ppm, however, the number of implantations was lower than controls by -15% due to a slightly higher pre-implantation loss of 12.2%, compared with the control value of 3.0%. There was no effect on the number of corpora lutea, implantations or on pre-implantation loss at either 2500 or 7000 ppm.
Post-implantation loss at 15000 ppm was higher at 31.2% compared with a control value of 4.0%, resulting in a lower number of pups being born per litter (8.8 compared to the control value of 14.6).
At 2500 and 7000 ppm, although post-implantation loss was slightly higher than controls at 8.4% and 11.5% respectively, the numbers of implantation sites and pups born for these groups was within the range of biological variation (for historical control data see Attachment "HCD_Gestation Day21" under Attached Background Material). Therefore, it was considered that there was no effect on post-implantation loss at up to 7000 ppm.
For details see Attachment "Summary of P female pre- and post-implantation loss" under Attached Background Material.

LITTER SIZE AND PUP MORTALITY
At 15000 ppm, the mean number of pups born was 8.8 which was 40% lower than the control value. The range was 2 to 14 pups born/litter at 15000 ppm with 15 dams having litters containing less than 12 pups, compared to 12 to 18 pups born/litter for controls.
Pup viability on PND 0 to 4 and PND 4 to 21 was comparable to controls at all concentration levels.

One male pup at 15000 ppm was born with a deformed hind limb and was culled on PND 4; the hind limb was short with no paw and there were only two digits defined.

The ratio of male to female pups was similar to controls at all chloroethane concentrations administered.

For details see Attachment "Summary of P female mating performance_fertility_duration of gestation_litter performance" under Attached Background Material.


OVARIAN SCORING
The examination of the ovaries from the control and high dose (15000 ppm) groups (quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea) found no test item-related effects and none of the data was statistically significant (some of the ovarian sections were incomplete and unsuitable for assessment and therefore it was not possible to evaluate all of the ovarian follicles sections prepared).
For details see Attachment "Summary of P female ovarian scoring" under Attached Background Material.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEC
Remarks:
general toxicity
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects noted up to and including 15000 ppm
Key result
Dose descriptor:
NOAEC
Remarks:
reproductive toxicity
Effect level:
7 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (sperm measures)
reproductive performance

Target system / organ toxicity (P0)

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
15 000 ppm
System:
male reproductive system
Treatment related:
yes
Dose response relationship:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
15 000 ppm
System:
female reproductive system
Treatment related:
yes
Dose response relationship:
yes

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At up to 15000 ppm, the clinical observations recorded were considered not to be test item-related as they were of low incidence, or were also seen in some control animals or were not concentration level-related.
The clinical observations of fur staining and/or wet fur were considered to be related to the administration procedure
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No test item-related mortalities were noted in F1 animals.

One male mid dose pup was accidentally killed during litter observation procedures when it was dropped on the floor.
A total of four Cohort 1A animals (2 low dose, 1 control and one high dose) were euthanised on Dosing Day 2, 9, 30 and 3, respectively, due to accidental tail damage incurred during the inhalation procedure. Grossly, these animals had swelling, open wounds/missing skin or a scab at the tip of the tail which correlated microscopically with mild acute subcutaneous haemorrhage and marked ulceration (i.e. skin loss) of the tail, in keeping with the reported clinical history of a traumatic tail injury. These deaths were considered not to be test item-related.
On Dosing Day 65, one high dose Cohort 1B male had repeated convulsions during the day and was euthanised for welfare reasons. Grossly, there were dark red foci in the right caudal and accessory lung lobes, most likely consistent with agonal congestion/haemorrhage. There were no additional gross lesions, and other tissues from this animal were not examined microscopically. No underlying cause of the convulsions was determined but this was considered unlikely to be test item-related, as convulsions are occasionally seen in rats.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
On PND 4, the litter weight (total weight of all live pups before culling) at 15000 ppm was 28% lower than the control value due to the lower number of pups born at 15000 ppm. This is considered to be attributable to the lower litter size in the group. Mean litter weight at 2500 and 7000 ppm was comparable to controls.
At 15000 ppm from PND 1 to 7, mean pup weight for both sexes was higher than controls by up to 21% for males and up to 20% for females. On PND 14 and 21, mean pup weight was similar to controls at 15000 pm. Mean pup weight at 2500 and 7000 ppm was comparable to controls from PND 1 to 21.
For details see Attachment "Summary of F1 pup weights and body weight gains" under Attached Background Material.

In Cohort 1A males and females, there was no effect on body weight up to Nominal Day 57 at any chloroethane concentration level administered.
For Cohort 1B males given 15000 ppm, there was a trend of slightly lower weight gain, with on Nominal Day 71 body weight gain being 7% less than controls (not statistically significant). There was no effect on body weight gain on Nominal Day 71 for Cohort 1B males given 2500 or 7000 ppm or females given up to 15000 ppm.
For details see Attachment "Summary of F1 body weights and body weight gains" under Attached Background Material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
For Cohort 1A and 1B males, there was no notable effect on food consumption up to Nominal Day 57 at any chloroethane concentration administered, although, there were occasional low values, compared with controls, for Cohort 1B males at 15000 ppm (-10% on Nominal Day 22/29 p≤0.05, -9% on Nominal Day 36/43).
For Cohort 1B females given 15000 ppm on Nominal Day 1/8, food consumption was -11% lower than controls (p≤0.01). For Cohort 1A and 1B females, there were occasional low values in test groups compared with controls, however, the magnitude of difference from controls was not concentration level-related.
For details see Attachment "Summary of F1 animal food consumption" under Attached Background Material.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No intergroup differences were noted by visual inspection of water bottles.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related haematology or coagulation changes were noted at any chloroethane concentration level administered in samples taken on the morning of necropsy for the F1A animals.
All differences in haematology or coagulation parameters were considered not to be test item-related based on their small magnitude, inconsistent direction, absence of a dose response, general overlap of individual values with the range of control and/or were of a magnitude of variation commonly observed in rats.
For details see Attachment "Summary of F1A animal haematology and coagulation data" under Attached Background Material.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No test item-related clinical chemistry changes were noted at any chloroethane concentration level in samples taken on the morning of necropsy for the F1A animals.
All differences in clinical chemistry parameters were considered not to be test item-related based on their small magnitude, inconsistent direction, absence of a dose response, general overlap of individual values with the range of control and/or were of a magnitude of variation commonly observed in rats.
For details see Attachment "Summary of F1A animal clinical chemistry data" under Attached Background Material.

THYROID HORMONES
There were no test item-related changes noted at up to 15000 ppm chloroethane for samples taken on the morning of necropsy for the F1A animals. Furthermore, there were no test item-related changes noted at up to 15000 ppm for samples taken from culled pups on PND 4 or unselected pups on PND 22 to 24.

All differences in these parameters were considered not to be test item-related based on their small magnitude, inconsistent direction, absence of a dose response, general overlap of individual values with the range of control and/or were of a magnitude of variation commonly observed in rats. For details see Attachment "Summary of F1 animal thyroid hormones" under Attached Background Material.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related effects on any urine parameter collected during the last week of dosing at up to 15000 ppm for the F1A animals.
All differences in urine parameters were considered not to be test item-related based on their small magnitude, inconsistent direction, absence of a dose response, general overlap of individual values with the range of control and/or were of a magnitude of variation commonly observed in rats.
Sexual maturation:
effects observed, treatment-related
Description (incidence and severity):
For both F1 generation male cohorts, there was a delay in sexual maturation of approximately 4 days at 7000 and 15000 ppm, compared with controls. The mean body weight at sexual maturity for these males was 10% to 17% higher than control values.

There was considered to be no effect on sexual maturity for males at 2500 ppm or for females at up to 15000 ppm, as the approximately 1 to 2 days difference from controls was considered to be within the range of biological variation.
For F1 females, there was no test item-related effect on the number of days recorded before an oestrus occurred, after vaginal opening.

For details see Attachment "Summary of F1 animal sexual maturation data" under Attached Background Material.
Anogenital distance (AGD):
effects observed, treatment-related
Description (incidence and severity):
At 15000 ppm on PND 1, male and female pups weighed 10% or 11% more respectively, than control pups. Pup body weight at 2500 or 7000 ppm was similar to control values.
For males at 7000 and 15000 ppm, absolute anogenital distance was slightly lower at 2.9 mm compared to 3.1 mm for controls. Normalised anogenital distance was slightly lower at 1.4 at 15000 ppm only, compared with 1.6 for controls. There was no test item-related effect on anogenital distance for male pups at 2500 ppm or for females at any chloroethane concentration level administered.
For details see Attachment "Summary of F1 animal anogenital distances" under Attached Background Material.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
On PND 13, there was no effect on nipple retention in male pups i.e. no nipples were present, at concentration levels up to 15000 ppm.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Cohort F1A animals (PND 84-94)
Test item-related organ weight differences were present in the thyroid gland, kidney and liver.
In F1A males at ≥ 7000 ppm, mean absolute and body weight adjusted thyroid gland and kidney weights were higher than controls.
In F1A females at ≥ 2500 ppm, mean absolute and body weight adjusted liver weights were higher, at ≥ 7000 ppm mean absolute thyroid gland weights were higher and at 15000 ppm mean absolute and body weight adjusted kidney weights were higher, when compared to controls.
There were additional organ weight values that were different from their respective controls. There were, however, no patterns or correlating data to suggest these values were related to the administration of chloroethane.
Test item-related organ weight differences are summarised in Table 9.

Cohort F1B animals (PND 94, PND 95, PND 98-100)
Test item-related organ weight differences were present in the thyroid gland, kidney and liver.
In F1B males at ≥ 2500 ppm, mean body weight adjusted liver weights were higher and at 15000 ppm, mean body weight adjusted thyroid gland and kidney weights were higher than controls.
In F1B females at 15000 ppm, mean body weight adjusted liver weight was also higher than controls.
The lower mean absolute pituitary gland weight and higher mean body weight adjusted testis weight in males at 15000 ppm were considered to be associated with the lower mean terminal body weight in this group.
There were additional organ weight values that were different from their respective controls. There were, however, no patterns or correlating to suggest these values were related to the administration of chloroethane
Test item-related organ weight differences are summarised in Table 10.

Non-selected F1 weanlings (PND 22-24)
There were no test item-related organ weight differences.
There were marked differences in mean absolute liver, spleen and thymus weight in both sexes when compared to controls (particularly in males). These tissues were not examined microscopically. However, given the lack of similar patterns (indeed liver weights were actually lower than controls, in contrast to the increases seen in F0 and F1 animals) and the absence of correlating histological findings in these organs in F0, F1A and F1B animals there was considered to be insufficient evidence to support a treatment-related effect in non-selected F1 weanlings.


Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort F1A (PND 84-94)
There were no test item-related gross findings.
Prostatic masses (similar to those seen in P animals above) were present in 2 control males and pale discolouration and an abnormally firm consistency of the prostate gland was reported in one high dose male. These findings in the prostate gland and all other gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be related to the administration of chloroethane.

Cohort F1A (PND 94, PND 95, PND 98-100)
There were no test item-related gross findings.
Similar to those lesions seen in the prostate gland of P and F1A cohort animals, a prostatic mass was evident in one control male and pale discolouration with/without abnormally firm consistency of the prostate gland was reported in one low dose and one mid dose male. These tissues were not examined microscopically (as per protocol) but given the similarity to lesions examined previously in other cohorts and the small number of animals affected (including controls and low and intermediate dose groups only), these findings were not considered to be test item-related along with all the other gross findings observed which were of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be related to the administration of chloroethane.

Pups Culled (PND 4)
There were no test item-related gross findings.
One high dose male pup had a deformed hindlimb; it was shortened with no paw and only two defined digits. The remaining pups had no gross abnormalities at necropsy. Given that only a single pup was affected, this deformity was considered not to be test item-related.

Non-selected F1 Weanlings (PND 22-24)
The right eye was missing from one mid dose female pup. Considering that this was the only pup affected and there were no gross abnormalities in any of the remaining pups, this was considered not to be test item-related.

For details see Attachment "Summary of F1 animal gross pathological data" under Attached Background Material.

Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Cohort F1A
There were no test item-related microscopic findings. There were however, microscopic findings of note in the prostate gland which were not considered to be test item-related.
Similar to that seen previously in F0 animals, the grossly evident masses present in the prostate gland of two control males and the pale discolouration and abnormally firm consistency of the prostate of one high dose male, correlated microscopically with moderate to marked, acute to chronic active inflammation; there was no histological evidence of neoplasia. As such, these lesions were considered not to be test item-related.
All the other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals, and, therefore, were also considered not to be test item-related.

Cohort F1B
Tissues from these animals were not examined microscopically as guideline and study plan.

Non-selected F1 Weanlings
Only the mammary gland was examined microscopically. Despite examination of best possible sections, mammary gland was only available for microscopic examination in 2/9 males and 5/11 females receiving 15000 ppm and 6/10 male and 9/10 female controls. There were no microscopic abnormalities evident in the tissue examined to support the presence of a test item-related effect and the small number of samples likely reflects the young age and small size of animals in this group, however, accurate interpretation is difficult given the small number of samples examined (especially in males).
The small number of remaining protocol-required tissues that could not be examined in F0 and F1 Cohort 1A animals were spread throughout the treatment groups and therefore were considered not to have impacted the quality or integrity of the study.

Ovarian Scoring
The examination of the ovaries from the control and high dose (15000 ppm) groups (quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea) found no test item-related effects and none of the data was statistically significant (some of the ovarian sections were incomplete and unsuitable for assessment and therefore it was not possible to evaluate all of the ovarian follicles sections prepared).
For details see Attachment "Summary of F1 female ovarian scoring" under Attached Background Material.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Sperm Analysis:
There were no notable test item-related effects on the percentage of motile sperm or straight line velocity at up to 15000 ppm. The percentage of progressively motile sperm was -41% lower than controls at 15000 ppm with a greater number of males (9/20) having progressive motility of less than 10% (controls 2/20). One mid dose male had a very low sperm motility value (33%).

At 15000 ppm, the total number of sperm was slightly lower than control by -17%. Values at 2500 and 7000 ppm were comparable with the control mean. There were no notable effects on sperm morphology at the concentrations examined of 7000 and 15000 ppm.

There was no test item-related effect on the number of spermatids for Cohort 1A males.

For details see Attachment "Summary of F1 male sperm measures" under Attached Background Material.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
There were no test-item related or dose dependent responses observed at necropsy of F1A animals for any of the immune cell populations analysed. The relative percentages of all immune cell populations analysed were comparable with the control animals.
For details Attachment "Summary of F1 animal immunophenotyping data" under Attached Background Material.

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEC
Remarks:
developmental toxicity
Generation:
F1
Effect level:
15 000 ppm
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects noted up to and including 15000 ppm
Key result
Dose descriptor:
NOAEC
Remarks:
developmental toxicity
Generation:
F1
Effect level:
2 500 ppm
Based on:
test mat.
Sex:
male
Basis for effect level:
sexual maturation
other: reduced anogenital distance at ≥ 7000 ppm, reduced total number of sperm and percentage progressively motile at 15000 ppm

Target system / organ toxicity (F1)

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
7 000 ppm
System:
male reproductive system
Treatment related:
yes
Dose response relationship:
yes

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
7 000 ppm
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes

Any other information on results incl. tables

Table 4. P Animals - Atmosphere Concentration Group Mean Values

F0 Animals

Dose Group/

Treatment

Study Days

Atmosphere Concentration
(ppm)

Difference from Target

(± %)

Target

Achieved

1

Air Control

1-127

0

0.00 (-)

0

2

Low Dose

1 -127

2500

2480 (13.9)

-0.9

3a

High Dose

1-2

7000

17200 (4.9)

+145.2

3-6

15000

14100 (3.3)

-5.7

7-18

20000

17100 (26.9)

-14.6

23-129

15000

15200 (13.4)

+1.5

4b

Intermediate Dose

1-2

15000

20800 (8.7)

+38.4

7-129

7000

6870 (12.8)

-1.9

Values in parentheses are % RSD
aFor Day 1-2 of dosing of the F0 generation, Group 4 target dose level was 15000 ppm to allow acclimatisation to the test item, based on results from a previous study.

bFor Day 3-6 of dosing of the F0 generation, Group 3 target dose level was 15000 ppm to allow acclimatisation to the test item based on results from a previous study.

cGroup 4 was not dosed on Days 3-6 but the pre-mating period was extended to achieve 10 weeks of dosing

dGroup 3 were not dosed on Days 19-22 but the pre-mating period was extended to achieve 10 weeks of dosing

Table 5. F1 Animals - Atmosphere Concentration Group Mean Values

F1 Animals

Dose Group/

Treatment

Study Days

Atmosphere Concentration
(ppm)

Difference from Target

(± %)

Target

Achieved

1

Air Control

122-199

0

0.00 (-)

0

2

Low Dose

115-196

2500

2530 (19.7)

+1.2

3

High Dose

119-199

15000

14400 (8.9)

-3.9

4

Intermediate Dose

119-199

7000

7310 (6.4)

+4.4

Values in parentheses are % RSD

Table 6. P and F1 Animals - Atmosphere Concentration Group Mean Values

F0 and F1 Animals

Dose Group/

Treatment

Study Days

Atmosphere Concentration
(ppm)

Difference from Target

(± %)

Target

Achieved

1

Air Control

All

0

0.00 (-)

0

2

Low Dose

All

2500

2500 (16.8)

+0.2

3b

High Dose

1-2

7000

17200 (4.9)

+145.2

3-6

15000

14100 (3.3)

-5.7

7-18d

20000

17100 (26.9)

-14.6

23-199

15000

14900 (12.2)

-0.4

4a

Intermediate Dose

1-2c

15000

20800 (8.7)

+38.4

7-199

7000

7040 (11.2)

+0.6

Values in parentheses are %RSD

aFor Day 1-2 of dosing of the F0 generation, Group 4 target dose level was 15000 ppm to allow acclimatisation to the test item, based on results from a previous study.

bFor Day 3-6 of dosing of the F0 generation, Group 3 target dose level was 15000 ppm to allow acclimatisation to the test item based on results from a previous study.

cGroup 4 was not dosed on Days 3-6

dGroup 3 were not dosed on Days 19-22

Table 7. Dosing Holidays

Dose

Group/Level

Animal Number

Not Dosed on Day(s)

Reason For Dosing Holiday

F0 Generation

1/0 ppm

M1001

58-59

Laceration on ventral neck

2/2500 ppm

M2002

72-73, 79-80

Limping on left hind limb

2/2500 ppm

M2015

29

Injured toe

2/2500 ppm

M2021

120

Injured hind toe nails

F1 Generation Cohort 1A

1/0 ppm

F1618

PND 50-51, 53-56

Wet lesion on dorsal neck

4/7000 ppm

F4615

PND 22

Tail damage (dosed for 72 min only)

F1 Generation Cohort 1B

2/2500 ppm

M2219

PND 66-67 and 88

Scabbed, bleeding lesion on neck (dosed for 163 min on PND 66 and 243 min on PND 88)

3/15000 ppm

F3201

PND 85

Weight loss due to damaged teeth

3/15000 ppm

F3208

PND 86

Convulsions (euthanised on PND 86)

PND – postnatal day

 

Table 8. Summary Group Mean Organ Weight Data – P Animals

 

Males

Females

Group

1

2

4

3

1

2

4

3

Dose (ppm)

0

2500

7000

15000

0

2500

7000

15000

No. animals examined

25

25

25

24

23

25

20

18

Terminal Body Weight (g)

523.5

505.8

525.4

494.5

286.1

272.5

282.1

281.7

Gland Thyroid

(No. weighed)

(25)

(25)

(25)

(24)

(23)

(25)

(20)

(18)

Absolute value

0.02404

0.02456

0.02663

0.02759**

0.02255

0.02108

0.02337

0.02190

% of body weight

0.00463

0.00494

0.00512

0.00558**

0.00793

0.00773

0.00824

0.00776

Kidney

(No. weighed)

(25)

(25)

(25)

(24)

(23)

(25)

(20)

(18)

Absolute value

3.7032

3.5405

3.6442

3.8991

2.1932

2.1555

2.3517*

2.3901**

% of body weight

0.71015

0.70451

0.69515

0.78968**

0.76870

0.79230

0.83317**

0.84219**

Liver

(No. weighed)

(25)

(25)

(25)

(24)

(23)

(25)

(20)

(18)

Absolute value

16.6973

16.0875

16.7702

17.9243

11.7298

11.6953

13.4249**

14.3583**

% of body weight

3.17920

3.18237

3.18076

3.62787**

4.08881

4.29310

4.73912**

5.08436**

Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

Table 9. Summary Group Mean Organ Weight Data – F1A Animals

 

Males

Females

Group

1

2

4

3

1

2

4

3

Dose (ppm)

0

2500

7000

15000

0

2500

7000

15000

No. animals examined

20

19

20

20

19

20

20

20

Terminal Body Weight (g)

356.8

342.2

384.2

347.9

214.8

216.5

220.2

220.0

Gland Thyroid (No. weighed)

(20)

(19)

(20)

(20)

(19)

(20)

(20)

(20)

Absolute value

0.01661

0.01676

0.02709++

0.02391++

0.01705

0.01573

0.02096+

0.02094+

% of body weight

0.00468

0.00475

0.00714++

0.00687++

0.00794

0.00729

0.00958

0.00949

Kidney

(No. weighed)

(20)

(19)

(20)

(20)

(19)

(20)

(20)

(20)

Absolute value

2.5947

2.6259

2.8457*

2.7670

1.5623

1.5404

1.6416

1.7197 *

% of body weight

0.72631

0.74044

0.74809

0.79667**

0.71914

0.71403

0.74516

0.78256**

Liver

(No. weighed)

(20)

(19)

(20)

(20)

(19)

(20)

(20)

(20)

Absolute value

12.9995

13.5294

14.3789

13.2075

7.4621

8.2716

8.4499 *

8.9955 **

% of body weight

3.64237

3.78871

3.76830

3.78710

3.42957

3.81103**

3.82855**

4.09385**

Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

Kruskal-Wallis & Dunn:+= p ≤ 0.05;++= p ≤ 0.01

Table 10. Summary Group Mean Organ Weight Data – F1B Animals

 

Males

Females

Group

1

2

4

3

1

2

4

3

Dose (ppm)

0

2500

7000

15000

0

2500

7000

15000

No. animals examined

20

20

20

19

20

20

20

20

Terminal Body

Weight (g)

390.2

392.3

407.0

367.4

232.9

234.0

233.4

228.3

Gland Thyroid

 (No. weighed)

(16)

(20)

(6)

(7)

(19)

(20)

(6)

(6)

Absolute value

0.01744

0.01803

0.01763

0.02004

0.01844

0.01691

0.01618

0.01737

% of body weight

0.00449

0.00461

0.00467

0.00583*

0.00794

0.00726

0.00688

0.00780

Kidney

(No. weighed)

(20)

(20)

(20)

(19)

(20)

(20)

(20)

(20)

Absolute value

2.7811

2.7540

2.9227

2.8627

1.6858

1.6238

1.7241

1.7191

% of body weight

0.71379

0.70140

0.71840

0.78487**

0.72471

0.69495

0.73880

0.75394

Liver

(No. weighed)

(20)

(20)

(20)

(19)

(20)

(20)

(20)

(20)

Absolute value

14.9535

16.1158

16.9523

15.5860

9.3195

9.4587

9.8437

10.0672

% of body weight

3.83157

4.10003*

4.16349**

4.26595**

4.00889

4.03844

4.21599

4.40355**

Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

Applicant's summary and conclusion

Conclusions:
Chloroethane administration to Sprague-Dawley rats by snout only inhalation for 6 hours daily, 7 days/week at concentrations up to 15000 ppm for 18 weeks to F0 males, and 10 weeks prior to mating, through mating, gestation and lactation to F0 females, and from PND 21 to at least PND 84 to F1 animals, was tolerated with no adverse clinical findings. At 15000 ppm, males had slightly lower body weight gain associated with slightly lower food consumption and during gestation and/or lactation, females had a lower body weight gain at ≥2500 ppm with slightly lower food consumption noted mainly at 15000 ppm. For F0 females pre- and post-implantation loss were higher by 12.2% and 31.2%, respectively, at 15000 ppm, and 40% less pups were born than for controls. On PND 1 at 15000 ppm, pups weighed more and for male pups, anogenital distance was slightly lower. For F1 males, there was a 4 day delay in sexual maturation at 7000 and 15000 ppm, although the pup mean body weight was higher than controls. For F0 and F1 males, the total number of sperm and percentage progressively motile were slightly lower at 15000 ppm. There were higher thyroid gland, kidney and/or liver weights in F0 and/or F1 animals at ≥2500 ppm but as there were no histopathological findings in these organs, the changes were considered not to be adverse.
The NOAEC (No Observed Adverse Effect Concentration) for F0 adult toxicity was considered to be 15000 ppm as the in-life or pathological findings were considered not to be adverse. The NOAEC for F0 male and female reproduction parameters was considered to be 7000 ppm due to the slight shift in the duration of gestation, effects on sperm numbers, progressive motility and pre- and post-implantation loss at 15000 ppm chloroethane. For F1 developmental parameters, the NOAEC was considered to be 2500 ppm due to the slightly lower anogenital distance and delay in sexual maturation for males at ≥7000 ppm.