1-[difluoro(trifluoromethoxy)methoxy]-1,2,2-trifluoroethylene

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

Combined oral repeated dose inhalation toxicity study and reproduction/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 December 2015 (arrival of test substance) to 21 September 2017 (final report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

28 July 2015

Deviations:

yes

Remarks:

Minor deviations not considered to have affected the validity and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

{difluoro[(1,2,2-trifluoroethenyl)oxy]methoxy}trifluoromethane

EC Number:

615-064-0

Cas Number:

700874-87-9

Molecular formula:

C4F8O2

IUPAC Name:

{difluoro[(1,2,2-trifluoroethenyl)oxy]methoxy}trifluoromethane

Test material form:

liquid: volatile

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar Han IGS rats (Crl:WI(Han))
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 or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: female rats were about 10 weeks old and the male rats about 9 weeks old at arrival. Females were 12 weeks old and males were 11 weeks old at the start of pre-treatment period. The age difference between males and females is deliberate, to avoid mating of siblings.
- Weight at study initiation: Body weight at allocation were within ± 20% of the mean weight for each sex.
On day 0 of treatment (pre-mating), mean BW were:
For Males: Controls: 347.87 +/- 15.62 g, Low dose: 346.73 +/- 15.82 g, Mid-dose: 344.18 +/- 17.74 g, High dose: 344.68 +/- 17.38 g.
For Females: 221.66 +/- 11.24 g, Low dose: 222.36 +/- 6.75 g, Mid-dose: 219.93 +/- 8.13 g, High dose: 224.00 +/- 8.34 g.
- Fasting period before study: No
- Housing: Makrolon cages, after allocation, during pre-mating period, the animals were housed 4/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. Animals were identified by a subcutaneous transponder. In addition, trail marks were used for additional identification within the cage
- Diet : ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days prior to the start of exposure.

DETAILS OF FOOD AND WATER QUALITY:
- Food: cereal-based (closed formula) powder rodent diet (VRF1(FG)). Each batch of this diet is analyzed by the supplier for nutrients and contaminants. The certificate of analysis pertaining to the batches used during the study (batches 2016 and 2372) are available.
- water: 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, chemical and microbiological examination of drinking water as conducted by the supplier are made available to the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C.
- Humidity (%): 30-70%
- Air changes (per hr): 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: From: 11-May-2016 (arrival of the animals) To: 4-Aug-2016 (necropsy of dams and pups)

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nose-only inhalation chambers (Institute’s design) consisting of a cylindrical PVC column with a volume of 75 litres,
surrounded by a transparent hood.
- Method of holding animals in test chamber: The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column.
- Source and rate of air: The total flow through the unit was at least 1 liter/min per rat.
- Method of conditioning air: a positive pressure was applied in the central column and a slightly negative pressure in the outer hood which encloses the entire animal holder, so dilution of the test atmosphere by air leaking from the animals’ thorax to the nose was avoided.

- System of generating vapours: To generate the test atmosphere, the test material cylinder was heated using a water bath to approximately 25°C (slightly above the test material’s boiling point) and a controlled amount of liquid test material was allowed to evaporate in a mass flow controlled stream (Bronkhorst Hi Tec, Ruurlo, The Netherlands) of humidified compressed air. The resulting gaseous atmosphere was led through a condense trap, diluted in a mass flow controlled stream (Bronkhorst Hi Tec) of humidified compressed air, and passed through a U-tube evaporator (kept in generation system after the preceding range finding study, but served no particular purpose during the present study). This main stream of test atmosphere was subdivided using rotameters and further diluted using mass flow controlled streams of humidified compressed air, which were subsequently directed to the inlets of the exposure chambers and led towards the noses of the animals. The test atmospheres were exhausted at the top of the exposure units.
The T95 was calculated to be between 6.8 and 7.6 minutes. The animals were placed in the exposure unit after stabilization of the test atmosphere, at least 8 min after the start of the test atmosphere generation.
The exposure chamber for the control animals (group 1) was supplied with a mass flow controlled stream (Bronkhorst Hi Tec) of humidified compressed air only.

- Temperature, humidity, pressure in air chamber: details in Attachment 1
- Air flow rate: average total air flows (+/- SD) : details in Attachment 1
- Air change rate: at least 1 liter/min per rat
- 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: Total carbon analysis. The response of the analyzers was recorded on a PC every minute using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses of the analyzers were converted to concentrations by means of calibration graphs (the formulas used to convert responses into concentrations are given below). For each exposure day, the mean concentration was calculated from the values determined every minute.
Detection: flame ionization detector, calibrated prior to the start of the study.
- Samples taken from breathing zone: yes. 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.

VEHICLE (if applicable)
- Composition of vehicle: clean air
- Oxygen concentration during exposure: 20.0 - 20.7 % (v/v)
- Carbon dioxide concentration: 0.073 - 0.756 % (v/v)

Analytical verification of doses or concentrations:

yes

Remarks:

Total carbon analysis

Details on analytical verification of doses or concentrations:

The actual concentration of the test material in the test atmospheres was measured by total carbon analysis,following a calibration study.
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 (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses of the analyzers were converted to concentrations by means of calibration graphs. For each exposure day, the mean concentration was calculated from the values determined every minute.

Calibration:
The output of the flame ionization detector of the TCA was calibrated using gas sample bags.
For the high concentration range (group 4), sample bags were filled with an accurate (mass flow controlled) volume of air and an accurate (weighed) amount of test material, injected into the bag through a septum. Three concentrations were thus prepared (in duplicate) – at approximately 80%, 100% and 120% of the target concentration of group – and analyzed by the TCA. The TCA’s of the low and mid concentration groups were calibrated by extracting mass flow controlled streams of atmosphere from the sample bags prepared at the high concentration, which were subsequently diluted in mass flow controlled streams of air. The resulting atmospheres, prepared (in duplicate) at approximately 80%, 100% and 120% of the low and mid target concentrations, were used to calibrate the analyzers of groups 2 and 3. Prior to calibration of the TCA’s, the mass flow controllers (Bronkhorst Hi Tec, Ruurlo, The Netherlands) used for dilution of the atmosphere were calibrated using a volumetric flow meter (DryCal, Bios International Corporation, Butler, NJ, USA). Linear relations were found between the response of the analyzers and the concentration of the test material (see below).
The calibrations were checked weekly during the study. To this end, atmospheres were prepared close to each target concentration using (dilutions of) sample bags as described above, which were subsequently analysed by the TCA. If the measured concentration deviated more than 5% from the calculated concentration, the calibration check was repeated. If the deviation was more than 5% at the re-check, a complete re-calibration was carried out.

Duration of treatment / exposure:

nose-only inhalation during 6 hours per exposure day.

Total exposure duration (see details under "Frequency of treatment"):
- Male animals were sacrificed after 29 days of exposure.
- Female animals were sacrificed on day 13 of lactation or shortly thereafter. (total number of exposures: approximately 40-47 days)

Frequency of treatment:

- Pre-mating period: males and females, during 2 weeks, 5 days/week (a total of 10 exposures)
- Mating period: males and females, daily until mating occurred.
- Post-mating period: males and non-mated females were exposed daily until sacrifice.
- Gestation period: daily exposure of mated females from evidence of mating until gestation day 19 (included)
- Lactation period: daily exposure from lactation day 5 until lactation day 12 (included)
Mated females were not exposed to the test substance bewteen gestation day 19 and lactation day 4 to allow the females to litter.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/L air (nominal)

Doses / concentrations 2

Dose / conc.:

1 mg/L air (nominal)

Doses / concentrations 3

Dose / conc.:

1.01 mg/L air (analytical)

Remarks:

SD: +/- 0.09

Doses / concentrations 4

Dose / conc.:

3 mg/L air (nominal)

Doses / concentrations 5

Dose / conc.:

3.02 mg/L air (analytical)

Remarks:

SD: +/- 0.07

Doses / concentrations 6

Dose / conc.:

12 mg/L air (nominal)

Doses / concentrations 7

Dose / conc.:

12 mg/L air (analytical)

Remarks:

SD: +/- 0.2

No. of animals per sex per dose:

12 males and 12 females per group

Control animals:

yes

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The exposure levels were selected on the basis of the results of a 14-day dose range finding study with the test substance in rats, and acute inhalation toxicity data.
- Rationale for animal assignment: computer randomization proportionally to body weight and taking into account the estrus cyclicity of the females.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily observation in the morning hours

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily observation in the morning hours, and halfway through the 6-hour exposure period, in particular to monitor any breathing abnormalities and restlessness.

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/ animal/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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on Day 14 of treatment, prior to the end of the premating period, by orbita punction whilst under sodium pentobarbital anaesthesia
- Anaesthetic used for blood collection: Yes (Na pentobarbital anaesthesia)
- Animals fasted: Yes, fasted overnight (water available)
- How many animals: 5/sex/group
- Parameters listed below "under Any other information on materials and method" were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on Day 14 of treatment, prior to the end of the premating period, by orbita punction whilst under sodium pentobarbital anaesthesia ; Because of clotting of the majority of the blood samples, another sampling was done at necropsy (day 33 relative to start date) from the 5 males and 5 females per group selected for extended histopathology. The later was used for the evaluation.
- Animals fasted: Yes, fasted overnight (water available)
- How many animals: 5/sex/group
- Parameters listed below "under Any other information on materials and method" were examined.

URINALYSIS: Not examined

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: detailed clinical examinations in an arena outside the home cage were performed on all rats of all groups prior to the first exposure and then once weekly until sacrifice for males, or until delivery of the first lot for females. Arena testing was also conducted prior to sacrifice (last week of the study) in the females concerned, as part of the Functional Observational Battery.
- Dose groups that were examined: all rats from all groups for detailed clinical examinations; For FOB: 5 males/group and 5 females/group with a litter
- Battery of functions tested:
FOB included assessment of the following functional domains :
* autonomic (lacrimation, salivation, pupil response to light, palpebral closure, piloerection, defecation, urination)
* neuromuscular (gait, mobility, forelimb and hindlimb gripstrength, landing foot splay, righting reflex)
* sensorimotor (response to tail pinch, click, touch and approach of a visual object)
* convulsive (clonic and tonic movements)
* excitability (ease of removal, handling reactivity, arousal, vocalizations)
* activity (rearing, posture)
* physiological (body temperature).
Motor activity was assessed following FOB testing in open roofed cages.

IMMUNOLOGY: Not examined

OTHER:
- Estrus cycle: 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.

Sacrifice and pathology:

- Male animals were sacrificed after 29 days of exposure.
- Female animals were sacrificed on day 13 of lactation or shortly thereafter. (approximately day 52-55 from study start)
- 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 PATHOLOGY: Yes (see table 1 - attachment 2)

HISTOPATHOLOGY: Yes (see table 1 - attachment 2)

Other examinations:

Fertility parameters in males and females: reported under IUCLID section 7.8.1 - Toxicity to reproduction

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.
Hormone levels (T4, TSH) in serum were determined with commercially available ELISA kit.

* 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).

* Nipple retention in male pups (lactation day 13)
On post-natal day 4, litter size was adjusted by eliminating extra pups by random selection to yield as nearly as possible 4 pups/sex/litter, preferentially at least 4 male pups/litter to assess nipple retention examination on Lactation day 13.
On postnatal day 13 all surviving male pups were examined for the presence of nipples or areolas.

* pup thyroid weight (at sacrifice, on lactation day 13)

Statistics:

Tests were generally be performed as two sided tests with results taken as significant where the probability of the results was 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.
- FOB: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor activity data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks (each session consists of 5 time blocks of 6 min each).
- Clinical pathology data (hematology, clinical chemistry): ‘Generalised Anova/Ancova q Test’ with ‘Automatic’ as data transformation method. This test was an automatic decision tree consisting of:
(1) Data preprocessing tests. First, normality of data distribution (Shapiro-Wilks test) and homogeneity of variances (Levene test) were checked (initial transformation ‘None’ [Identity]). If any of these checks failed (p<0.05) they were repeated using Log transformation. If checks on log-transformed data failed, data were rank-transformed
(2) A group test assessing whether or not group means were 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 were 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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One female of the low dose group was sacrificed moribund during delivery. Clinical signs included piloerection, hunched posture, general pale and general cold observations. Upon necropsy, the fetuses were dead inside the uterus with fetuses in the birth canal. No gross lesions were observed that could explain the moribund condition. The main finding at microscopical examination was multifocal moderate hepatocellular necrosis in the liver. Although the origin of this histopathological change remains unclear, it was probably responsible for the moribund condition of the animal. In absence of a dose relationship this finding was considered not to be related to treatment.
The other clinical observations were mainly related to the skin and considered normal for this strain of rats.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

See details above, one female of the low dose group was sacrificed moribund during delivery. Based on the macroscopical and histopathological findings, and lack of dose relationship, the moribund condition of the animal was considered not to be related to treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related or statistically significant changes in red blood cell or clotting variables in either males or females.
The relative lymphocyte count was statistically significantly decreased in females of the high dose group. As the absolute lymphocyte count was not affected, this was not ascribed to treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In males, data from blood samples taken on day 33 of the study showed a statistically significant decrease in ASAT levels in the males of all treatment groups, and a statistically significant decrease in ALAT levels was observed in the males of the mid and high concentration groups, compared to the control group.
The ASAT concentrations in controls were above the historical control data. As the measured values of ASAT in the treated groups were within the historical control data and in absence of a dose-response relationship this was not considered to be related to treatment.
Because the ALAT concentration measured in the control animals was higher than the historical control data, and in absence of a dose-response relationship, these observations are not considered related to treatment.

A statistically significantly increase in bilirubin concentrations was observed in the males of the mid and high concentration groups.
In absence of a dose-response relationship and because the concentration measured in the control animals was lower than the historical control data, these observations are not considered related to treatment.
A statistically significantly increase in PO4 concentrations in the mid-concentration group females was considered a chance finding in absence of a dose-response relationship.
A statistically significant increase in bile acids in the high dose group males was considered treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no statistically significant organ weight changes in males of any treated group compared to controls, including for the reproductive organs.
In females of the mid- and high-concentration group the absolute uterus weight was statistically significantly decreased. In absence of a dose response relationship and in absence of effects on the relative uterus weight, this was considered not relevant.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy no treatment related macroscopic changes were observed in the surviving animals.
In the female of the low dose group that was found moribund, no gross lesions were observed that could explain the moribund condition.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The only remarkable treatment-related findings were in levels 3, 4, 5 and 6 of the nose, predominantly in the dorsal meatus, and consisted in minimal (12/12 males, 11/12 females) to mild (1/12 female) degeneration of the olfactory epithelium: disarrangement of the nuclei, increased vacuolation and decreased size of the apical part of the olfactory epithelial cells. These histopathological changes were observed in all males and all females of the high dose group only.
No other histopathological changes were considered treatment-related as they occured equally in different groups, or in few animals and were common findings in rats of this strain and age.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

* Estrus cycle:
In the low dose group, a statistically significant number of females showed acyclicity during the pre-mating period. In the absence of dose-response this was not ascribed to treatment.
There were no treatment-related effects on cyclicity, length of the longest cycle or mean length of the longest cycle. (see details in Table)

* Hormone analysis in males F0, in male pups and female pups (F1) sacrificed at lactation day 13: No significant differences were observed for the concentrations of TSH and T4 in the sera from exposed animals as compared to the control animals.
Clear differences were found for the TSH and T4 concentrations between the adult F0 (males) samples and the samples of F1 pups (males and females) sacrificed at lactation day 13 (in relation to age-difference).

* Fertility and reproductive performance: (see details in table and in IUCLID section 8.7.1)
- No effect on fertility, the mating index was 100% in all treatment groups and 91.7% in the control group.
There was no treatment-related differences in the length of gestation. There was no stillborn pups.

* Litter data: see Table attached.
The number of pups born 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 indeces (4-d survival and 13-d survival) were not affected by the treatment.

* Pup anogenital distance: no differences were observed when corrected for body weight.
* Pup nipple retention: no effects were observed in the male pups
* Pup body weight and organ weight: no treatment-related effects observed on terminal body weight or thyroid weight.
* Pup macroscopic examination: no observed effects.

Effect levels

Key result

Dose descriptor:

NOEC

Effect level:

3.02 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

12 mg/L air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

other: olfactory epthelium

Treatment related:

yes

Dose response relationship:

yes

Applicant's summary and conclusion

Conclusions:

Following the 29 days of inhalation exposure in males and 40-47 days for females, based on the effects on the histopathological changes in the nose and the statistical significant increase in bile acids in the males of the high concentration group, the No Observed Effect Concentration (NOEC) for parental toxicity was determined to be at 3 mg/L (mid concentration).
In absence of effects on fertility parameters, reproductive performance and development, the NOEC for reproduction and developmental effects was placed at 12 mg/L (the highest concentration tested).

Executive summary:

The test substance was administered by inhalation (6 hours/day) at concentrations of 0 (control), 1, 3 and 12 mg/L 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 29 days for males and 40-47 days for females.

There was no treatment-related mortality. Daily clinical observations did not reveal any treatment-related clinical signs. Neurobehavioral observations and motor activity assessment did not indicate any neurotoxic potential.

There were no relevant changes in body weight, food intake, red blood cell variables and clotting potential. Clinical chemistry showed a statistically significant increase in the concentration bile acids in the males of the high concentration group. No effects were observed on organ weight or at macroscopic examination.

In the high concentration group only, histopathological examination showed treatment related changes in the dorsal meatus in slides from several levels in the nose (levels 3, 4, 5, and 6) in all males and females. The changes comprised minimal (12 males, 11 females) to mild (1 female) degeneration of the olfactory epithelium, characterized by disarrangement of the nuclei, increased vacuolation and decreased size of the apical part of the olfactory epithelial cells. The effects as observed in the high concentration group appeared not to be present in the mid and low concentration groups.

There were no other significant findings.

There were no effects of the test substance on male and female fertility, reproductive performance, litter data, pup survival or pup development.

There were no effects on hormone levels between the exposed groups and the control groups for male adults and there were no effects on hormone levels between the exposed groups and the control groups for 13-day old male and female pups.

Based on the effects on the histopathological changes in the nose and the statistical significant increase in bile acids in the males of the high concentration group, the No Observed Effect Concentration (NOEC) for parental toxicity was determined at 3 mg/L (mid concentration) (3.02 mg/L analytical concentration).

In absence of effects on fertility parameters, reproductive performance and development, the NOEC for reproduction and developmental effects was placed at 12 mg/L (the highest concentration tested).