Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7-SEP-2016 to 21-NOV 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
SOURCE OF TEST MATERIAL
- batch No.of test material: SMF271SG08 to SMF271SG17 (same production batch supplied in 10 cylinders)
- Expiration date of the lot/batch: 11-08-2021
Cross-reference
Reason / purpose:
reference to same study
Remarks:
satellite study of OECD421 screening
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7-SEP-2016 to 21-NOV 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Only minor deviations not considered to have affected the validity of the study
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SMF271SG08 to 17 (same production batch dispatched in 10 individual 1L cylinders)
- Expiration date of the lot/batch: 11/08/2021
- Purity test date: 11/08/2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient temperature
- Stability under test conditions: stable
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han IGS (Crl:WI(Han))
Details on species / strain selection:
This rat strain is routinely used at the test facility for this type of studies (available historical control database)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulsfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: at arrival females were about 10 weeks old and males were about 9 weeks old; Females were 12 weeks old and males were 11 weeks old at the start of pre-treatment period, 14 weeks (F) / 13 weeks (M) at the start of exposure, and 16 weeks (F) / 15 weeks (M) at the start of mating.
The age difference between males and females is deliberate, to avoid mating of siblings.
- Weight at study initiation:
On day 0 of treatment (pre-mating), mean BW were:
For males: controls: 342.17 +/- 14.54 g, low dose: 339.52 +/- 14.28 g, mid-dose: 335.67 +/- 14.67 g, high dose: 337.07 +/- 13.98 g
For females: controls: 224.65 +/- 7.97 g, low dose: 223.86 +/- 5.20 g, mid-dose: 225.43 +/- 6.84 g, high dose: 226.08 +/- 8.39 g

- Fasting period before study: not applicable
- Housing: Makrolon cages, after allocation, during pre-mating period, the animals were housed 4 or 5/cage (separated by sex). For mating, one male and one female were housed together. Mated females were house individually. During exposure periods, the rats were individually housed in the exposure unit without access to food or water. Immediately after exposure, the animals were returned to their home cage.

- Diet : ad libitum. Cereal-based (closed formula) powder rodent diet (VRF1(FG)). Each batch of this diet is analyzed by the supplier for nutrients and contaminants.
- Water : ad libitum. domestic mains tap water suitable for human consumption. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chem ical and microbiological examination of drinking water as conducted by the supplier are made available to the test facility
- Acclimation period: at least 14 days prior to the start of exposure

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: From: 7-SEP-2016 (arrival of the animals) To: 22-NOV-2016 (necropsy of dams and pups)
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
clean air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 2.2 m3 whole body exposure units (based on the design by Hazleton Systems, Inc., Aberdeen, MD, USA). These chambers were constructed of stainless steel, with glass doors on two sides which allowed observation of the animals during exposure. The test atmosphere was introduced at the top and exhausted at the bottom of the chamber
- Method of holding animals in test chamber: During exposure, animals were housed individually in Type II Macrolon cages.
- Source and rate of air: The ventilation rate was at least 10 air changes per hour.
- Method of conditioning air: The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere.
- System of generating test atmosphere: The test atmospheres were generated by mixing a controlled stream of gaseous test material with a controlled flow of dry pressurized air. For safety reasons, this was performed in an airtight chamber. The resulting test atmosphere was mixed with the main air stream (available as a laboratory provided source of non-pressurized, HEPA-filtered air), which was subsequently directed to the inlets at the top of the exposure chamber; the atmosphere was exhausted at the bottom. The exposure unit for the negative control animals was supplied with a controlled flow of HEPA-filtered air only.

The T95 (time to reach the steady state concentration), based on chamber volume and total air flow range was calculated to be between 13 and 21 minutes. The animals were placed in the exposure chamber prior to the start of the test atmosphere generation. Test atmosphere generation was stopped six hours after the start of generation. The animals were removed from the exposure chamber after the concentration had dropped below a level of 1% of the target concentration.

- Temperature, humidity, pressure in air chamber: Temperature of 22 ± 3ºC ; relative humidity between 30 and 70%
- Air flow rate: Initially, the flow of test material was controlled using mass flow controllers (Bronkhorst HiTec, Ruurlo, The Netherlands). During the course of the first week of exposure however, the mass flow controllers became increasingly difficult to regulate, because the test material seemed to affect the inside valves. Thus, from 14 October 2016 onwards, the mass flow controllers were replaced by peristaltic pumps (Watson-Marlow type 502s, Bredel Pumps Limited, Falmouth, Cornwall, England).
The flow of test atmosphere was controlled using a constant volume controller and was measured in the exhaust of the exposure chamber using a KIMO air velocity sensor (type CTV110-AOD150; KIMO, Emerainville, France). The air flow was continuously measured and recorded on a PC every minute using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany).
Prior to the first exposure of the animals, homogeneous distribution of the test material in the exposure chambers was confirmed by analysis of samples taken at five different locations in each exposure chamber (deviation of individual values from the mean of all five samples should not exceed 10%; the actual maximum deviation was 4.6% for the low-concentration group).

- Air change rate: at least 10 air changes per hour.
- Treatment of exhaust air: The exhausted air of the inhalation exposure unit is connected to a ventilation duct (at negative pressure). The exhausted test atmosphere flow is mixed with ambient air from the outer cylinder (hood) of the exposure unit. The resulting diluted test atmosphere is mixed with test atmosphere (or clean air) from other groups, filtered and by means of a ventilator exhausted outside the building.

TEST ATMOSPHERE
- Brief description of analytical method used: the actual concentrations were analysed by photoacoustic infrared for groups 2 and 3, or total carbon analysis for group 4.
- Samples taken from breathing zone: yes

VEHICLE (if applicable)
- Composition of vehicle: clean air
- Oxygen concentration during exposure: 20.6 - 20.8 % (v/v)
- Carbon dioxide concentration: 0.079 - 0.116 % (v/v)
Details on mating procedure:
- M/F ratio per cage: 1 male and 1 females per cage during the mating period
- Length of cohabitation: all mating couples had sperm-positive confirmation of mating within 4 mating days, except for 1 female in the control group
- Proof of pregnancy: presence of sperm in vaginal smear was considered gestation day 0.
- Further matings after two unsuccessful attempts: not necessary
- After successful mating each pregnant female was caged : individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
* Low and mid-concentration groups:
The actual concentration of the test material in the low- (groups 2) and mid- (groups 3) concentration test atmospheres was measured by photoacoustic infrared analysis. Test atmosphere samples were analyzed intermittently, with an automatic switch between both chambers every five minutes using a valve system and a digital timer (Omron H5CX). The response of the analyzers was recorded on a PC every minute. The responses of the analyzers were converted to concentrations by means of calibration graphs.

Calibration:
Prior to the first exposure, the output of the photoacoustic infrared analyzer was calibrated using gas sample bags filled with (mass flow controlled) volumes of clean dry air, which were injected with known volumes of test material. For both target concentrations three calibration concentrations were prepared (approximately 80, 100 and 120% of the target concentration), at least in duplicate, and analyzed. The response (Y) of the analyzer was linearly related to the concentration (X, in ppm) of the test material as:
Y = 1.195 * X + 0.1878, with a coefficient of determination (R2) of 0.9983.
This relation was used to calculate the test atmosphere concentrations for groups 2 and 3. The average concentration during exposure was corrected for the duration of the animals’ stay in the exposure chamber and the number of measurements during exposure. The calibration was checked weekly during the study by measuring a concentration close to the target concentration, prepared in a gas sample bag as described above. If the measured concentration deviated more than 5% from the theoretical concentration and this was confirmed with a second sample bag, the analyzer was to be recalibrated. However, the
weekly calibration checks did not necessitate recalibration.

* High concentration group:
The actual concentration of the test material in the high-concentration test atmosphere was measured by total carbon analysis. Test atmosphere samples were taken continuously from the exposure chamber at the animals’ breathing zone and were passed to the total carbon analyzer (TCA) through a sample line. The response of the analyzers was recorded on a PC every minute using a CAN transmitter. The responses were subsequently converted to concentrations by means of a calibration graph.

Calibration:
Prior to the first exposure, the output of the flame ionization detector of the TCA was calibrated using gas samples bags, prepared as described for low and mid-dose groups. The response (Y) of the analyzer was linearly related to the concentration (X, in ppm) of the test material as:
Y = 1.024 * X – 3.826 (R2 = 0.9949).
This relation was used to calculate the test atmosphere concentrations for the high concentration group. The average concentration during exposure was corrected for the duration of the animals’ stay in the exposure chamber and the number of measurements during exposure. The calibration was checked weekly during the study as described for groups 2 and 3. The weekly calibration checks did not necessitate recalibration of the analyzer.

Generation efficiency:
Since the test material affected the mass flow controllers which were initially used to control the flow of test material during atmosphere generation (and were replaced by peristaltic pump as of 14 October 2016), the settings of the mass flow controllers could not be used for calculation of the generation efficiency. Instead, the total amount of test material used, was calculated from the measured concentration and the total flow of test atmosphere through the three whole-body exposure chambers. This calculated use was compared against the actual (weighed) use, determined as the weight difference of the test material cylinder before and after use2, taking into account the amount of material used for calibration of the analyzers. The generation efficiency was calculated from the calculated and the actual (weighed) use of test material, as follows:
Efficiency = 100 * total calculated use of test material for groups 2, 3 and 4 / actual use

Due to technical limitations explained above, it was not possible to accurately determine the daily generation efficiency. Instead, generation efficiency was calculated over three consecutive periods of several days, by comparing the test material consumption as calculated from the actual concentrations and flows against the consumption as determined from the weight decrease of the test material cylinder. This resulted in overall generation efficiencies of 101.1%, 100.9% and 92.5%, respectively, i.e. close to a 100% efficiency, which is expected for test atmosphere generation from a gaseous test material.
Duration of treatment / exposure:
Whole-body inhalation during 6 hours per exposure day.

Total exposure duration (see details under "Frequency of treatment"):
- Male animals were sacrificed after 28 days of exposure.
- Female animals were sacrificed on day 13 of lactation or shortly thereafter. The total number of exposure days was 40-47 in females.
- Female animals that failed to mate or appeared not pregnant after mating were sacrificed at least 21 days after the last mating date or at least 25 days after the presumed mating date, respectively
Frequency of treatment:
Exposure to the test substance during the different study phases:
- Pre-exposure period: male and females were not exposed.
- Premating period: male and female animals were exposed during 2 weeks prior to mating to the test substance for 5 days/week (i.e. 10 exposure days in total)
- Mating period: male and female rats were exposed daily until confirmation of mating.
- Post-mating period: males and non-mated females were exposed daily until sacrifice.
- Gestation period: mated female animals were exposed daily from successful mating (i.e. the finding of a sperm positive vaginal smear considered gestation day 0) until gestation day 19 (included).
- Lactation period: Females were exposed daily starting lactation day 5 until lactation day 12 (included).
There was no exposure of females between GD19 and LD4 to allow the females to litter. Daily exposure was resumed on lactation day 5 up to day 13 of lactation.
Details on study schedule:
Age at mating of the mated animals in the study: 16 weeks for Females / 15 weeks for Males.
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
5 ppm (nominal)
Dose / conc.:
4.95 ppm (analytical)
Remarks:
SD: +/- 0.24 ; average exposure
Dose / conc.:
15 ppm (nominal)
Dose / conc.:
15.04 ppm (analytical)
Remarks:
SD: +/- 1.05 ; average exposure
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
50.69 ppm (analytical)
Remarks:
SD: +/- 2.16; average exposure
No. of animals per sex per dose:
12/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The exposure levels were selected on the basis of the results of a previous 28-day inhalation study in Sprague-Dawley rats.
- Rationale for animal assignment: computer randomization proportionally to body weight.
Positive control:
No positive control was used for the reproductive endpoints of the main inhalation assay.
Mitomycin C was used as positive control for the in vivo micronucleus assay performed in males at the end of the treatment period. (reported in IUCLID section 7.6.2 genetic toxicity in vivo)
Mitomycin C was administered i.p. at 1.5 mg/kg bw in a single dose to a satellite group of 5 additional males used as positive controls for Micronucleus analysis.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily observation in the morning hours, before the exposure.
- Cage side observations listed in Attachment 2 were included.
- Animals were also observed halfway through the 6-hour exposure period to monitor any breathing abnormalities and any restlessness. Animals were again observed post-exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: once during the acclimatization period, at initiation of treatment, then weekly (males, and females during the pre-mating and mating period). Mated females were weighed on days 0, 7, 14 and 20 during presumed gestation and on day 0, 4, 7 and 13 of lactation. Non-mated females were weighed once per week after the mating period. All the animals were weighed on their scheduled necropsy date.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Food was refreshed once a week. The food consumption was measured per cage when body weight was measured, except during the mating period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on day of necropsy, blood will be sampled from aorta. For males: on day 30, for females: on day 51-53 (relative to study start).
- Anaesthetic used for blood collection: Yes (sodium pentobarbital anaesthesia)
- Animals fasted: Yes, fasted overnight (water available)
- How many animals: 5/sex/group
- Parameters:
Haemoglobin (Hb),
packed cell volume (PCV),
red blood cell count (RBC),
reticulocytes,
total white blood cell count (WBC),
differential white blood cell counts (neutrophils, lymphocytes, eosinophils, basophils, monocytes),
prothrombin time,
thrombocyte count.

The following parameters were calculated:
mean corpuscular volume (MCV),
mean corpuscular haemoglobin (MCH),
mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on day of necropsy. For males: on day 30, for females: on day 51-53.
- Animals fasted: Yes, fasted overnight (water available)
- How many animals: 5/sex/group
- Parameters:
alkaline phosphatase activity (ALP)
aspartate aminotransferase activity (ASAT) (glutamic-oxalacetic transaminase (GOT))
alanine aminotransferase activity (ALAT) (glutamic pyruvic transaminase (GPT))
gamma glutamyl transferase activity (GGT)
total protein
albumin
ratio albumin to globulin (calculated)
urea
creatinine
glucose (fasting)
creatinine kinase (CK)
bilirubin (total)
cholesterol (total)
triglycerides
phospholipids
calcium (Ca)
sodium (Na)
potassium (K)
chloride (Cl)
inorganic phosphate (PO4)
bile acids
Oestrous cyclicity (parental animals):
Vaginal smears were made daily in all females from the start of the pre-treatment period until confirmation of mating. An additional smear was made at the day of sacrifice.
Sperm parameters (parental animals):
Not examined
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- litters were reduced to a maximum of 4 pups/sex/litter with preferentially at least 4 male pups/litter to assess nipple retention examination on Lactation day 13; excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups, stillbirths, live births, dead pups, runts (pups that are smaller than control pups), postnatal mortality, presence of gross anomalies, evaluated on days 0 and 4, 7 and 13 of lactation.
- The alive pups were individually weighed on days 0 and 4, 7 and 13 of lactation. Mean pup weight was calculated per sex and for both
sexes combined.
- anogenital distance (AGD), in pups on Lactation day 4: The anogenital distance (AGD) was measured for each pup before culling of the litter. The AGD is reported corrected for body weight (cube root of the body weight).
- presence of nipples/areolae in all surviving male pups on lactation day 13


GROSS EXAMINATION OF DEAD PUPS:
yes, necropsy was performed on stillborn pups and pups dying during the study; macroscopic abnormalities were recorded.

OTHER: HORMONE ANALYSIS
The following parameters are part of the revised Guideline OECD422 (July 2015) to detect potential endocrine effects
* Blood sampling for hormone determinations: TSH and T4
- at necropsy, blood from all males and females was taken under i.p. sodium pentobarbital anaesthesia.
- on lactation day 4, blood sample collected from surplus pups per litter at culling. Blood was pooled.
- on lactation day 13, from 2 pups per litter (one male and one female), blood was collected from the heart under CO2/O2 anaesthesia during necropsy on or shortly after day 13.

* Anogenital distance in pups (Lactation day 4):
The anogenital distance (AGD) was measured for each pup before culling of the litter. The AGD is reported corrected for body weight (cube root of the body weight).

* pup thyroid weight (at sacrifice, on lactation day 13)
Postmortem examinations (parental animals):
SACRIFICE
- Male animals were sacrificed after 28 days of exposure.
- Maternal animals: Female animals were sacrificed on day 14 of lactation or shortly thereafter.
On the day of necropsy of all females a vaginal smear is taken to determine the stage of the estrous cycle as part of the histopathological examination of the reproductive organs.
Female animals that failed to mate or appeared not pregnant after mating were sacrificed at least 21 days after the last mating date or at least 25 days after the presumed mating date, respectively.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Attachment 2 were weighed and prepared for microscopic examination.
In addition, reproductive organs (ovaries, uterus, testes, epididymides, seminal vesicles and prostate) of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined.
Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at lactation day 13.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- At necropsy of the dams and litter, at or shortly after day 13 of lactation, pups were examined externally for gross abnormalities and sacrificed by gradual CO2 anesthesia.

HISTOPATHOLOGY / ORGAN WEIGHTS
The thyroid gland was sampled from 2 selected pups per litter, weighed and preserved for histopathological examination (not examined in this study).

HORMONE ANALYSIS (T4, TSH):
- in serum from F1 pups on day 13, from 1 male and 1 female pup/litter. Analysis performed with commercially available ELISA kit, and according to a validated method.
In addition, blood samples were collected from all surplus pups per litter at culling on lactation day 4. (not analysed in this study)
Statistics:
Tests were generally performed as two-sided tests with results taken as significant where the probability of the results is p<0.05 (*) or p<0.01 (**).
- Continuous data were subjected to a decision tree for continuous data
- Dichotomous data were evaluated using a decision tree for dichotomous data.
- Clinical pathology data (haematology, clinical chemistry): ‘Generalised Anova/Ancova q Test’ (abbreviation GEN AN) with ‘Automatic’ as data transformation method (abbreviation AUTO). This test is an automatic decision tree consisting of:
(1) Data preprocessing tests. First, normality of data distribution (Shapiro-Wilks test) and homogeneity of variances (Levene test) are checked (initial transformation ‘None’ [Identity]). If any of these checks fail (p<0.05) they are repeated using Log transformation. If checks on log transformed data fail, data are rank-transformed
(2) A group test assessing whether or not group means are all equal (parametric for untransformed or log-transformed data: one-way analysis of variance [Anova]; non-parametric for rank transformed data: Kruskal-Wallis test).
(3) Post-hoc analysis. If the group test shows significant (p<0.05) nonhomogeneity of group means, pairwise comparisons with the control group are conducted by Dunnett’s multiple comparison test (parametric after Anova, non-parametric after Kruskal-Wallis; significance levels 0.01 and 0.05).

- Incidences of histopathological changes: Fisher’s exact probability test.
Reproductive indices:
Reproductive indices measured
- number of females mated (= placed with males)
- number of females inseminated
- number of males with pregnant females
- duration of gestation (mean number of gestation days)
- number of females surviving delivery
- number of females with live born and (all) stillborn pups
- number of implantation sites
- number of corpora lutea and preimplantation loss: not determined in this study since the dams are sacrificed at lactation day 13, making the determination difficult.

reproduction indices (calculated):
. mating days until Day 0 pc = time between the start of mating and successful copulation
. duration of gestation = time between gestation day 0 and day of delivery
. female mating index = (number of females inseminated/number of females placed with males)x 100
. male mating index = (number of males placed with females /number of females inseminated)x 100
. female fertility index = number of pregnant females*100/number of inseminated females
. male fertility index = number of males with pregnant females*100/number of males placed with females
. gestation index = (number of females with live pups / number of females pregnant) x 100
. prenatal loss = (Total number of Implantations - Total number of pups delivered) x 100 / Total number of Implantation Sites
. perinatal loss = (Total number of pups delivered - Total number of alive pups delivered)x 100 / Total number of pups delivered
Offspring viability indices:
- litter size: number of pups delivered (live- and stillborn) per litter
- number of live pups at day 0 and 4, and on day 7, and 13
- number of pups lost
. live birth index = (number of pups born alive/number of pups born) x 100
. viability index day 0 - 4 = (number of pup surviving 4 days/number of liveborn on day 0) x100
. sex ratio day 0 = [(number of live male or female pups on day 0 / number of live pups on day 0] x100
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A lower mean body weight (5%), but non-statistically significant, was observed in males of the mid and high concentration groups at the end of the exposure period.
A transient decrease in mean body weight gain in the males in the mid and high concentration groups, concurrent to a transient decrease in mean food consumption in the males in the high concentration group, which was limited to the first two weeks of exposure (premating phase).
No treatment related effects were observed in body weight in females (despite generally lower body weight gain in treated females during pre-mating phase compared to the control group, with no difference in food consumtion). Body weight change was statistically lower in the first week of gestation in females of the mid and high concentration groups, compared to the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
A transient decrease in mean food consumption was observed in the males in the high concentration group, which was limited to the first two weeks of exposure (premating period).
No treatment related effects were observed in food consumption in females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
No statistically significant changes were observed in red blood cell parameters and in prothrombin time (males, females).
In males, a statistically significant lower absolute number of neutrophils in the low concentration group was considered a chance finding in absence of a concentration response relationship.
In females, a statistically significant increase in the percentage lymphocytes was observed at the mid and high concentration groups, and a decrease in the percentage neutrophils. In absence of statistically significant differences in absolute lymphocytes and neutrophils this was considered not toxicologically relevant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Glucose plasma levels were increased in males in the low and high concentration groups. However, in the absence of dose-dependence and no similar findings in females, the effect was not considered related to treatment.
A statistically significant decreased mean creatinine levels in females in the high concentration group were considered to be related to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
An increased relative mean relative kidney weight was observed in males in the mid and high concentration group. The mean absolute kidney weight did not reach statistical significance.
In females absolute and relative mean kidney weight were increased in the high concentration group. In addition mean relative lung weight was increased in the females in the high concentration group, without concentration dependent relationship.
No effects were observed on absolute and relative reproductive organ weights.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic evaluation did not reveal treatment related histopathological changes. The increase in absolute and relative kidney weights in the high concentration Females and the increase in relative kidney weights in the mid and high concentration males were statistically significantly higher than in controls. However, this could not be explained by microscopical findings in the kidneys.
In general, the histopathological changes observed were about equally distributed amongst the different treatment groups or occurred in one or a few animals only. They are common findings in rats of this strain and age or occurred as individual chance findings. Therefore, they were not considered to be related to treatment.

All animals in the high concentration group showed stripy pattern on the teeth, which is considered to be related to the fluor in the test substance. Animals in the intermediate groups were not examined.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Reproductive parameters:
Mating occured within 4 days in all treatment groups, except one female in the control group. All mated females were pregnant except 1 female (#55) in the mid concentration group. All pregnant females delivered litters with liveborn pups. One litter (#53) of the mid concentration group had 7 stillborn pups and 1 live pup.
Key result
Dose descriptor:
NOAEC
Effect level:
15 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: general toxicity
Key result
Dose descriptor:
NOEC
Effect level:
>= 50 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Fertility, reproductive parameters
Critical effects observed:
yes
System:
urinary
Organ:
kidney
Treatment related:
yes
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
No specific difference in pup survival, pre and postnatal loss between treated groups and controls.
Body weight and weight changes:
effects observed, treatment-related
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:
no effects observed
Description (incidence and severity):
No statistically significant difference in males and female absolute and relative thyroid weight.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Description (incidence and severity):
No findings in thyroid gland from pups aged 13 days.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
There was no effect on TSH levels measured on lactation day 13 in male and female pups.
In female pups, the T4 levels were statistically significantly increased in the mid-concentration group. However, in the absence of a concentration related response and because the value was within historical control data, it was considered not related to treatment.

Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
* Litter data: The number of pups per litter and the sex ratio were comparable in the various groups. Live birth index (% live born pups) was not affected by the treatment (100% in all groups) and viability indices (4-d survival and 13-d survival) were not affected by the treatment.
* Pup anogenital distance on day 4: no differences were observed in males and female pups when corrected for body weight.
* Pup nipple retention: There was no difference in the mean number of (rudimental) nipples per group.
* Pup body weight: Mean pup weight was statistically significantly lower for male pups in the high concentration group on day 0 and day 4, as well as in the low concentration group on day 4. Mean pup weight was statistically significantly lower for females in the low and high concentration groups, on day 0, day 4, day 7 and day 13.
* Pup body weight gain: there was no statistically significant difference between groups for the pup body weight gain during the period PND0 to PND13.
* Pup thyroid weight: no statistically significant difference in male and female pups examined on lactation day 13.
* Hormone analysis: There was no effect on TSH levels measured on lactation day 13 in male and female pups.
In female pups, the T4 levels were statistically significantly increased in the mid-concentration group. However, in the absence of a concentration related response and because the value was within historical control data, it was considered not related to treatment. Thus, it was concluded there was no effect on thyroid hormone (T4) and TSH levels measured on lactation day 13 in male and female pups.
* Clinical effects: there were no treatment-related clinical observations in pups
* Pup macroscopic examination: no observed effects.
Key result
Dose descriptor:
NOAEC
Generation:
F1
Effect level:
15 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Reproductive effects observed:
no
Conclusions:
Under the conditions of the study, the No Observed Adverse Effect concentration (NOAEC) for maternal systemic toxicity was 15 ppm hexafluorobutadiene, based on the effects on absolute and relative kidney weight, relative lung weight and mean creatinine level in females in the high concentration group (50 ppm).
Based on the effects on relative kidney weight in males in the mid and high concentration group and the transient effects on body weight and food consumption in the first two weeks of exposure in males in the mid and high concentration groups, the No Observed Effect Concentration (NOEC) was placed at the low concentration (5 ppm hexafluorobutadiene).
In the absence of effects on fertility and reproductive parameters, the NOEC for reproduction was 50 ppm, the highest concentration tested.
Based on a slightly lower mean pup weight in the high concentration group, the NOAEC for developmental toxicity was placed at the mid concentration (15 ppm hexafluorobutadiene)
Executive summary:

The possible effects of hexafluorobutadiene on reproductive performance and pup development were investigated in a screening study (Test guideline OECD421) conducted in Wistar rats. The test substance was administered by inhalation (6 hours/day) at concentrations of 0 (control), 5, 15 and 50 ppm during a premating period of 2 weeks (5 days/week, 10 exposure days in total), then daily during mating and post-mating up to sacrifice for the males.

Females were exposed to the test atmospheres during a premating period of 2 weeks, during mating, gestation and lactation. Mated females were not exposed to the test substance between gestation day 19 and lactation day 4 in order to allow the females to litter. Daily exposure was resumed on lactation day 5 up to day 13 of lactation. The total number of exposure days was 28 days for males and 40-47 days for females.

 

The test atmospheres were stable and the actual concentrations were close to intended.

Exposure to 0, 5, 15 or 50 ppm hexafluorobutadiene resulted in:

No treatment-related mortality and morbidity.

A transient decrease in mean body weight gain in the males in the mid and high concentration groups and a transient decrease in mean food consumption in the males in the high concentration group, which was limited to the first two weeks of exposure.

No treatment related effects were observed in food consumption or body weight in females.

An increased relative mean relative kidney weight in males in the mid and high concentration group. In females absolute and relative mean kidney weight were increased in the high concentration group. In addition mean relative lung weight was increased in the females in the high concentration group.

All animals in the high concentration group showed stripy pattern on the teeth, which is considered to be related to the fluor in the test substance. Animals in the intermediate groups were not examined.

No other treatment-related findings were observed in histopathological examination.

No effects on red blood cell parameters, white blood cell parameters and prothrombin time.

A statistically significant decrease in mean creatinine level in females in the high concentration group.

No effects on fertility or reproduction parameters.

No effects on T4 and TSH hormone levels in adult males, and in male and female pups (lactation day 13).

No effects on pup sex and survival, pup observations, anogenital distance or nipple retention. Macroscopic and microscopic examination of the pup thyroid did not show any effects.

A slight decrease in mean pup weight in the high concentration group was considered related to treatment. No effects were observed on pup weight gain.

 

Based on the effects on absolute and relative kidney weight, relative lung weight and mean creatinine level in females in the high concentration group, the No Observed Adverse Effect concentration (NOAEC) for maternal toxicity was placed at the mid concentration (15 ppm hexafluorobutadiene).

Based on the effects on relative kidney weight in males in the mid and high concentration group and the transient effects on body weight and food consumption in the first two weeks of exposure in males in the mid and high concentration groups, the No Observed Effect Concentration (NOEC) was placed at the low concentration (5 ppm hexafluorobutadiene).

In the absence of effects on reproductive parameters, the No Observed Effect Concentration (NOEC) for reproductive toxicity/fertility was placed at the high concentration (50 ppm hexafluorobutadiene).

In absence of effects on pup sex and survival, pup observations, anogenital distance and nipple retention, but based on a slightly lower mean pup weight in the high concentration group, the No Observed Adverse Effect Concentration (NOAEC) for developmental toxicity was placed at the mid concentration (15 ppm hexafluorobutadiene).

 

Results of the bone marrow micronucleus test combined to the repeated exposure are reported under the in vivo genotoxicity section. Hexafluorobutadiene, at concentrations up to 50 ppm by inhalation, did not show any indication of chromosomal damage and/or damage to the mitotic spindle apparatus of the bone marrow target cells of male rats. Systemic availability of the test substance was demonstrated by slight effects on body weight change and food consumption in males, indicating that the negative response observed in this bone marrow micronucleus test is not due to lack of systemic availability of the test substance or its metabolites.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Not a stand-alone assay, but included as part of an OECD421 inhalation study with a 28-day treatment in males.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian cell cytogenetic assay

Test material

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

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han IGS (Crl:WI(Han))
Details on species / strain selection:
This rat strain is routinely used at the test facility for this type of studies (available historical control database)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS (mainly refering to males of the study)
- Source: Charles River Deutschland, Sulsfeld, Germany
- Age at study initiation: males were about 9 weeks old; males were 11 weeks old at the start of pre-treatment period, 13 weeks at the start of exposure.
- Weight at study initiation:
On day 0 of treatment, mean BW were:
controls: 342.17 +/- 14.54 g, low dose: 339.52 +/- 14.28 g, mid-dose: 335.67 +/- 14.67 g, high dose: 337.07 +/- 13.98 g

- Fasting period before study: not applicable
- Housing: Makrolon cages, after allocation, during pre-mating period, the animals were housed 4 or 5/cage (separated by sex). For mating, one male and one female were housed together. During exposure periods, the rats were individually housed in the exposure unit without access to food or water. Immediately after exposure, the animals were returned to their home cage.
- Diet : ad libitum. Cereal-based (closed formula) powder rodent diet (VRF1(FG)). Each batch of this diet is analyzed by the supplier for nutrients and contaminants.
- Water : ad libitum. domestic mains tap water suitable for human consumption. The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chem ical and microbiological examination of drinking water as conducted by the supplier are made available to the test facility
- Acclimation period: at least 14 days prior to the start of exposure

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 45-65%
- Air changes (per hr): 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

IN-LIFE DATES: From: 7-SEP-2016 (arrival of the animals) To: 2-NOV-2016 (necropsy of males)

Administration / exposure

Route of administration:
inhalation: gas
Vehicle:
clean air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The exposure conditions are reported under the OECD421 study.
Duration of treatment / exposure:
male rats: 28 days, 6hrs/day, 5 days/week
Frequency of treatment:
Exposure to the test substance during the different study phases:
- Pre exposure period: male were not exposed.
- Premating period: male were exposed during 2 weeks prior to mating to the test substance for 5 days/week (i.e. 10 exposure days in total)
- Mating period: male and female rats were exposed daily until confirmation of mating.
- Post-mating period: males were exposed daily until sacrifice for a total of 28 days.
Post exposure period:
The males were sacrificed the day following the last exposure
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
5 ppm (nominal)
Dose / conc.:
4.95 ppm (analytical)
Remarks:
SD: +/- 0.24 ; average exposure
Dose / conc.:
15 ppm (nominal)
Dose / conc.:
15.04 ppm (analytical)
Remarks:
SD: +/- 1.05 ; average exposure
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
50.69 ppm (analytical)
Remarks:
SD: +/- 2.16; average exposure
No. of animals per sex per dose:
5 males/concentration (one spare sample was collected from a sixth male rat in each group)
5 males in the positive control group
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Justification for choice of positive control(s): one of the standard positive controls recommended by the OECD474 guideline (there is no specific standard for inhalation route).
- Route of administration: intra-peritoneal
- Doses / concentrations: 1.5 mg/kg bw in physiological saline as the vehicle, administered about 24-hrs before sacrifice.
It is acceptable that the positive control be administered by a route different from the test chemical, using a different treatment schedule, and for sampling to occur only at a single time point.

Examinations

Tissues and cell types examined:
erythrocytes from the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- Dose selection rationale: The exposure levels were selected on the basis of the results of a 28-day inhalation study in rats. The micronucleus analysis is part of a reproduction/developmental screening study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
*Treatment:
Males were exposed for a total of 28 days.
For the micronucleus test, bone marrow was collected at scheduled necropsy, the day following the last treatment, from 5 males of each group (one spare sample was collected from a sixth male rat in each group; this sample was not evaluated).
In addition, bone marrow cells were collected from the 5 males in the positive control group (injected once with 1.5 mg/kg bw Mitomycin C, 24 hrs before sacrifice).

*Sampling:
Bone marrow cells from one of the femurs of the rats designated for the micronucleus test were immediately collected into foetal calf serum and processed into glassdrawn smears according to the method described by Schmid (1976).

DETAILS OF SLIDE PREPARATION:
Four bone marrow smears per animal were made, air-dried and fixed in methanol. Two fixed smears were stained with May-Grünwald Giemsa. The other two fixed smears were kept as reserve. If needed, these reserve slides were also stained with May-Grünwald Giemsa solution and used for microscopic examination.

METHOD OF ANALYSIS:
Randomly coded slides (two per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for examination are evenly distributed over the whole smear.

The following criteria were used for the scoring of cells:
• A polychromatic erythrocyte (PE) is an immature erythrocyte that still contains ribosomes and can be distinguished from mature, normochromatic erythrocytes by a faint blue stain.
• A normochromatic erythrocyte (NE) is a mature erythrocyte that lacks ribosomes and can be distinguished from immature, polychromatic erythrocytes by a yellow stain.
• A micronucleus is a small, normally round, nucleus with a diameter of circa 1/20 to 1/5 of an erythrocyte, distinguished from the cytoplasm by a dark blue stain.
Number of erythrocytes scored: at least 500 erythrocytes (E) per animal. The numbers of PE and NE were recorded.
If micronuclei are observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total of 500 E (PE + NE) have been scored, an additional number of PE were scored for the presence of micronuclei until a total of 4000 PE have been scored.
Evaluation criteria:
The micronucleus test was considered valid if the positive controls give a statistically significant increase in the mean number of MPE/4000 PE and the negative controls are within the historical range.
A substance was considered to cause chromosomal damage and/or damage to the mitotic apparatus if it showed a dose related positive response or a clear increase of micronucleated cells in a single dose group. A substance was considered to be negative in the micronucleus test if it produced no positive response at the dose level(s) analysed.
A response was considered to be positive if the mean number of MPE/4000 PE was statistically significantly higher compared to the negative control group.
Statistics:
In all tests a significance level of 5% was used (α = 0.05)
Data were analysed by one-way analysis of variance (ANOVA). Two ANOVA models were applied. In one of the ANOVA models it was tested if the positive control differed from the negative control (t test). In the other ANOVA model (including Dunnett’s test as post-hoc test) it was tested if the test substance (different doses) differed from the negative control. It was checked if the ANOVA assumptions were met (i.e. if variances are equal). In case assumptions were not met, square root transformation (sqrt(x+1)) was applied to 'normalise' the distribution of the counts. In case this would not be sufficient, non-parametric testing was
performed using the Mann-Whitney test (positive control compared with negative control) or Kruskal-Wallis analysis of variance (test material groups compared with negative control).

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS
- Clinical signs of toxicity in test animals: There was no clinical signs reported during the study period.

- Evidence of cytotoxicity in tissue analyzed: there was a slight concentration-dependent decrease in immature erythrocytes/total erythrocytes(-2.92%, -7.6%, -16.95% compared to the control group), although not statistically significant.

- Rationale for exposure: Systemic availability of the test substance was demonstrated by slight effects on body weight gain and food consumption in males, as well as on the relative kidney weight in the high concentration group. In addition, the blue stripy pattern noticed on the teeth of all the high concentration males shows plasmatic exposure to the test substance and/or its metabolites (e.g. fluoride or related metabolites) and resulting effect on enamel development.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
In the negative control group, the group mean number of micronucleated polychromatic erythrocytes (MPE) per 4000 polychromatic erythrocytes (PE) of the negative control group (clean air treatment) and the positive control group (treated intraperitoneally with 1.5 mg/kg bw mitomycin C) showed the expected response. Therefore the study met the validity criteria.

- Ratio of PCE/NCE (for Micronucleus assay):
- Frequency of micronucleated immature erythrocytes (PE): there was no difference in the treated groups compared to control.
- Frequency of micronucleated mature erythrocytes (NE): 0 in all groups treated for 28 days, as well as in the negative and positive control groups.

- Appropriateness of dose levels and route: Inhalation is the most likely route of exposure to the gaseous substance. The repeated exposure design is consistent with potential exposure in the industrial setting.

- Statistical evaluation:
There was no statistically significant increase in the mean number of MPE/4000 E or in the mean number of PE/500 E in any of the groups treated with Hexafluorobutadiene when comparing to the concurrent negative control group.

Any other information on results incl. tables

Mean numbers of PE/500 erythrocytes and MPE/4000 polychromatic erythrocytes

   test substance  target concentration

 PE/500 erythrocytes

(mean + SD)

MPE/4000 PE

(mean + SD) 

 control  clean air  -  171 + 32 4.6 + 2.2 
 low concentration Hexafluorobutadiene   5 ppm  166 + 26 4.0 + 1.4 
mid concentration Hexafluorobutadiene  15 ppm  158 + 43  5.8 + 2.4
high concentration Hexafluorobutadiene  50 ppm  142 + 21  4.0 + 1.9
 positive control MN  Mitomycin C  1.5 mg/kg bw i.p. (single dose)   93 + 23 (1)  65.8 + 8.3 (2)

(1)Unpaired t-test p-value: 0.0022

(2) Unpaired t-test p-value: < 0.0001 (after transformation of data)

Applicant's summary and conclusion

Conclusions:
There was no statistically significant increase in the mean number of micronucleated erythrocytes compared to the concurrent negative control animals. Under the conditions of the study, exposure to hexafluorobutadiene at up to 50 ppm by inhalation did not cause chromosomal damage to the bone marrow cells in male rats.
Executive summary:

In male rats exposed by inhalation for 28 days to 5, 15 or 50 ppm hexafluorobutadiene, bone marrow was collected from one of the femurs and processed to prepare 4 bone marrow smears per animal, that were air-dried and fixed in methanol, then stained. The slides were randomly coded and polychromatic erythrocytes (PE) and normochromatic erythrocytes (NE) were recorded in at least 500 erythrocytes per animal. Micronucleated erythrocytes (MPE or MNE) were scored until a total number of 4000 polychromatic erythrocytes was reached.

The group mean number of MPE per 4000 PE of the negative control group (group 1: clean air) and the positive control group (1.5 mg/kg-bw Mitomycin C) showed the expected responses. Therefore, the study was considered valid.

There was no statistically significant change in the mean number of micronucleated erythrocytes in a total of 4000 scored polychromatic erythrocytes and the mean number of polychromatic erythrocytes /500 erythrocytes in any treatment group compared to the negative control group.

Systemic availability of the test substance was demonstrated by slight effects on body weight change and food consumption, as well as by the blue pattern on the incisors indicating plasmatic exposure to the substance or fluoride derivative resulting in enamel change. There was a slight concentration dependent decrease in the ratio of immature polychromatic erythrocytes/total erythrocytes although not statistically significant.

It can be concluded that under the conditions of the study, the exposure to hexafluorobutadiene at up to 50 ppm for 28 days in male rats did not show evidence of chromosomal damage and/or damage to the mitotic spindle apparatus of the bone marrow cells.