Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with GLP and OECD guideline, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Triskelion bv, Ultrechtsweg 48, 3700 AJ Zeist, The Netherlands
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, the Netherlands
- Age at receipt 5-6 weeks
- Body weight at study initiation: Male: mean dose groups approx. 207 g; Female: mean dose groups approx. 171 g
- Housing: The animals were housed in macrolon cages with a bedding of wood shavings (Lignocel, Type ¾) and strips of paper (Enviro-dri) and wooden block as environmental enrichment. During premating animals were housed by sex, 4 to a cage, during mating 1 male plus 1 female, following mating females were housed individually and with their litter following delivery.
- Diet: Cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England). ad libitum, except during exposure
- Water: ad libitum except during exposure
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- temperature 22±2°C
- humidity: 45 - 65%
- air changes: 10/hr
- Photoperiod: 12 hrs dark/12 hrs light
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
other: combination nose only/ whole body
Vehicle:
air
Details on exposure:
EXPOSURE EQUIPMENT
Animals were exposed to the test atmosphere in head/nose-only exposure units (hereafter called nose-only exposure units) and by whole-body exposure chambers:
- Nose-only exposure: Each exposure unit (Institute’s design) consisted of a cylindrical PVC column with a volume of 75 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column, and was exhausted at the top. Each column included three rodent tube sections of 20 ports each. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column (males and females alternated). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. Animals were rotated each week with respect to the position in the column, viz. they were moved 5 places each time, and also weekly alternated between the upper, middle and lower sections. The units were illuminated externally by normal laboratory fluorescent tube lighting. The total air flow through the units was at least 1 L/min per animal. The air entering the units was controlled at 22 ± 3°C and the relative humidity was maintained between 30 and 70%.
- Whole-body exposure: Animals were exposed whole-body to the test atmosphere in 2.2 m3 whole-body exposure units (Hazleton Systems, Inc., Aberdeen, MD, USA). These chambers are 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 of the exposure chambers, and exhausted at the bottom. Each chamber could accommodate 28 cages to individually house the dams and 56 cages to individually house 28 male and 28 female animals up to approx. week 6. Animals rotated at least every week with respect to their position in the exposure chamber. The units were illuminated externally by normal laboratory fluorescent tube lighting. The ventilation rate was between 12 and 15 times/hour (corresponding to 440 – 550 L/min). The temperature in the chambers was controlled at 22 ± 3°C and the relative humidity was maintained between 30 and 70%.

GENERATION OF TEST ATMOSPHERE
- Nose-only exposure: The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. The cylinder containing the test substance was warmed using a heating jacket to pressurize the cylinder. To generate the test atmosphere, a liquid flow of test substance, controlled by a peristaltic pump (Watson-Marlow 502S, Bredel Pumps Limited, Falmouth, Cornwall, England), was evaporated in a stainless steel evaporator. The temperature of the evaporators was controlled at 20°C using a thermal bath. The vapor was transported in a stream of humidified compressed air, the flow of which was measured by a mass stream meter (Bronkhorst, Hi Tec, Ruurlo, The Netherlands), supplemented with a mass flow controlled stream of oxygen (mid and high concentration groups). The exposure unit for the control animals was supplied with a measured stream of humidified compressed air only. The generated test atmospheres were directed to the bottom inlets of the exposure units. At the top of the units the test atmospheres were exhausted. The animals were placed in the exposure units after stabilization of the test atmosphere.
- Whole-body exposure: The test atmosphere for each whole-body exposure chamber was generated similarly. Mass flow controlled amounts of gaseous test material were mixed with a main air stream, available as a laboratory provided source of (non-pressurized) filtered air. Oxygen was added for the mid and high concentration groups. To ensure a ventilation rate of 12-15 times/hour, air speed was measured continuously in the exhaust of the exposure chamber using a Ventcaptor air speed sensor (Weber Sensors Inc., Acworth, GA, USA). The flow was automatically adjusted to a predefined setpoint by a PID feedback system using an electrical valve (EA20, Georg Fischer AG, Schaffhausen, Switzerland). In addition, air speed in the exhaust of the exposure chambers was checked weekly using a rotary gas meter (IRM-A, Elster-Instromet, Luxembourg, Luxembourg).
Details on mating procedure:
At the end of the premating period, each female was caged with one male from the same group. Animals were caged together until mating occured or 1 week had elapsed. In the F1-generation the mating period was extended for one day. Mating pairs were clearly identified. Every consecutive morning during the mating period, vaginal smears were made for determination of the presence of sperm. The day on which sperm was detected in the vaginal smear was considered as gestation day 0. Upon evidence of copulation the females were caged individually for the birth and rearing of their pups. Sperm positive females that turned out to be non-pregnant were killed after more than 21 days after copulation. Females that did not show evidence of copulation after the end of the mating period were also housed individually until sacrifice (more than 21 days after the last day of the mating period). Dams were allowed to raise one litter.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The actual concentration of the test material in the test atmosphere was monitored with a total carbon analyser (for groups 2 and 3: Ratfisch RS55T, Munich, Germany; for group 4: Sick Instruments Benelux, Hedel, The Netherlands). The responses of the analysers were recorded on a PC every minute using a CAN transmitter (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany). Test atmosphere samples were taken continuously from the exposure unit at the animals’ breathing zone and were passed to the total carbon analyser through a sample line. The mean response was calculated by averaging values read every minute. Prior to the various exposures, the total carbon analysers were calibrated for a particular target concentration by sampling from three concentrations in duplicate in a range including the target concentration. The concentrations were prepared by injecting a known amount of 1233zd(E) into a PET sample bag containing a measured amount of air. These calibrations were checked weekly by measuring the concentration from a sample bag close to the target concentration. If the measured concentration from the sample bags deviated more than 5% from the calculated concentration during the study and this was confirmed with a second sample bag, the total carbon analyzer was calibrated again.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
F0 generation animals were exposed nose-only to the test material in air for 6 hrs/day/5 days/wk for at least 10 weeks prior to mating and daily during mating. Daily exposure continued for F0 females up to gestation day (GD) 19 for 6 hrs/day (nose only). From lactation day 5 (PN5), females were exposed daily (6 hrs/day) to the test substance by whole body exposure until the end of the lactation period (PN21) or their sacrifice shortly thereafter. The F1-generation male and female pups were exposed by whole body exposure (6 hrs/day, 5 days/wk) from postnatal day 22 up to ca. 6 wks of age. Subsequently, the F1 animals were exposed in a similar manner as that described for the F0 generation.
Non-mated females were exposed (nose-only) until the end of the nose-only exposure period. Non-pregnant animals were exposed by nose-only exposure until GD 19 of the presumed gestation period; thereafter the exposure was not resumed.
Details on study schedule:
Selection of parents from the F1 generation: On or shortly after PN day 21 the F1-pups were weaned and 28 males and 28 females were selected at random from as many litters as possible in each group to rear the next generation.
F1 parental animals not mated until at least 10 weeks after selection from the F1 litters.
Remarks:
Doses / Concentrations:
0, 2000, 5000, and 15000 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
28
Control animals:
yes, sham-exposed
Details on study design:
Exposure level selection rational: High exposure level same as used in 13-week inhalation toxicity study which resulted in heart lesions.

CULLING
On postnatal (PN) day 4, litters of more than 8 pups were adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 4 males and 4
females per litter. Pups euthanized at culling were examined externally for abnormalities and subsequently preserved in a neutral aqueous phosphate buffered
4% solution of formaldehyde.

WEANING AND SELECTION OF PUPS
On or shortly after PN day 21 the F1-pups were weaned and 28 males and 28 females were selected at random selection from as many litters as possible in each group to rear the next generation (see Annex 5). Furthermore, F1- and F2-generation pups were randomly selected for necropsy. Four spare pups per sex and dose group were selected at weaning of the F1-pups. From PN 22, these pups were exposed to the same concentrations as their mothers and
pups from the groups treated with the test substance were discarded on day 7 of the premating period of the F1-generation. Selected spare F1-pups of the control group were exposed until day 6 of the premating period (for possible replacement) and were sacrificed at the end of the in-life phase of the study (satellite animals).
Parental animals: Observations and examinations:
- General clinical observations: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days and on all exposure days, all cages were checked again in the afternoon for dead or moribund animals to minimize loss of animals from the study. In the period before the start of exposure on Saturdays, Sundays and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.
- Body weight: Body weights of male and female rats were recorded four days before the start of administration of the test substance at randomization, at the start of the study (day 0) and weekly thereafter during the premating period. Males were weighed once per week during the postmating period until sacrifice. Females were weighed once per week during postmating and mated females were weighed on days 1, 7, 14 and 21 during presumed gestation and on day 0, 4, 7, 14 and 21 of lactation. In addition, the animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.
- Food consumption: The food consumption was measured from day 0 onwards on the same days as the body weight was measured. Erroneously, no food consumption was measured of the not mated F1-females in the postmating period. The results are expressed in g per animal per day and g per kg body weight per day.
Oestrous cyclicity (parental animals):
Vaginal smears to evaluate the estrus cycle length and normality were made daily for about 3 weeks prior to mating. Smears were made and stained of all females, but only the smears of the control group (group 1) and the high-dose group (group 4) were evaluated. Since no treatment-related abnormalities were observed, the vaginal smears of the animals of the low- and mid-dose group were not examined.
Sperm parameters (parental animals):
- Epididymal sperm motility, count and morphology: At scheduled necropsy, epididymal sperm was derived from the left cauda epididymis of all males of all groups. For this purpose the cauda epididymis was dissected, weighed and thereafter minced in M199 medium containing 0.5% bovine serum albumin. Sperm motility and, after sonification and DNA staining, the cauda epididymal sperm reserves (sperm count) was measured for all males of all groups, using the Hamilton Thorne Integrated Visual Optical System (IVOS). In addition, a smear of the sperm solution was prepared and stained for all males, but only the smears of the control group (group 1) and the high-dose group (group 4) males were examined for morphology. No treatment-related changes were observed in the high-dose group, therefore the examination of sperm morphology was not extended to the low- and mid-dose groups.
- Testicular sperm count: At necropsy, the left testis of all males of all groups was placed on dry ice and subsequently stored in a freezer (<-70°C) for later determination of the number of homogenization-resistant spermatids. The testes to be analysed were thawed just before further processing. Following removal of the tunica albuginea, the testicular parenchyma was weighed, minced and homogenized in Saline Triton X-100 solution. Following DNA-staining, the homogenization-resistant sperm heads were enumerated using the IVOS and the daily sperm production was calculated. The evaluation of homogenization-resistant spermatids was performed in the control (group 1) and high-dose (group 4). No treatment-related changes were observed in the high-dose group, therefore the evaluation of homogenization-resistant spermatids was not extended to the low- and mid-dose groups.
Litter observations:
- Parturition and litter evaluation: At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.
- Litter size, sexes and weight: The total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups were evaluated on days 0, 4, 7, 14 and 21 of lactation. The pups were individually weighed on days 0, 4, 7, 14, and 21 of lactation. Mean pup weight was calculated per sex and for both sexes combined. The number of runts [defined as pup weight less than mean pup weight of the control group minus 2 standard deviations] was calculated.
- Sexual maturation: The following landmarks for sexual maturation were recorded in the selected F1-generation animals: males: balanopreputial separation from PN day 38 onwards; females: vaginal patency from PN day 30 onwards.
Postmortem examinations (parental animals):
- Gross necropsy and histology of parental animals: Males were sacrificed after successful mating and females were sacrificed at or shortly after weaning on PN day 21. All surviving parent male and female rats were euthanized by exsanguination from the abdominal aorta under CO2/O2 anaesthesia and then examined grossly for pathological changes. A necropsy was performed on animals that died intercurrently (if not precluded by autolysis) or that had to be killed because they were moribund. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde except for the testes which were preserved in Bouin's fixative: adrenals, brain, epididymides *(left cauda which was used for sperm analysis), heart*, kidneys, liver, ovaries including oviduct*, pituitary gland*, prostate*, seminal vesicles and coagulating glands*, spleen, testes*(left testis was used for sperm analysis), thyroid, uterus* (after counting of the implantation sites), vagina*, organs and tissues showing macroscopic abnormalities. All organs mentioned above except the vagina were weighed (paired organs together) as soon as possible after dissection to avoid drying. Tissues marked with an asterisk (*) were embedded in paraffin wax, sectioned at 5µm, and stained with haematoxylin and eosin, except for sections of the testes, which were stained with PAS (Periodic Acid Schiff). Microscopic examination was performed on these organs of all rats of the control and high-dose groups. As no treatment-related abnormalities were observed, the histopathological examination was not extended to the low- and mid-dose groups. In addition, reproductive organs 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.
Postmortem examinations (offspring):
- Gross necropsy and histology op pups and weanings: All stillborn pups and pups found dead were stored in a freezer (<-18°C) for macroscopical examination for structural and pathological changes. Gross necropsy was also performed on pups of dams that were sacrificed during lactation (these pups were sacrificed at the time of the dam’s death). Organs and tissues showing macroscopic abnormalities were preserved in a neutral aqueous phosphate buffered 4% solution of formaldehyde. After selection of the pups for the next generation, from the remaining pups 1 male and 1 female pup of each litter (as far as present) were subjected to a thorough necropsy. Pups were euthanized by exsanguination from the abdominal aorta under CO2/O2 anaesthesia and then examined grossly for pathological changes. Special attention was paid to the organs of the reproductive system. The following organs were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde: brain, spleen, thymus, organs and tissues showing macroscopic abnormalities. The brain, spleen, and thymus were weighed as soon as possible after dissection to avoid drying.
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 was subjected to a decision tree or parametric statistical test.
- Dichotomous data was evaluated using the statistical test Chi-square-Fisher.
- Incidences of microscopic observations in adult animals were evaluated by Fisher’s exact probability test.
Reproductive indices:
For each mating the following data was presented for each group:
- number of females mated (=number of females placed with males)
- number of males mated (= number of males placed with females)
- number of females inseminated (= number of successful copulations)
- number of females pregnant (demonstrated by the presence of implantation sites observed at necropsy)
- number of males with inseminated females
- number of males with pregnant females
- number of females with liveborn and (all) stillborn pups
- number of implantation sites
- number of corpora lutea
- number of pups delivered (live- and stillborn)
- number of lost pups
- mean number of litters with liveborn pups at day n

The following parameters were calculated:
- female mating index = number of females inseminated *100/number of females placed with males
- female fertility index = number of pregnant females*100/number of inseminated females
- male mating index = number of males with inseminated females*100/number of males placed with females
- male fertility index = number of males with pregnant females*100/number of males placed with females
- pre-coital time = mating days until day 0 post coitum (time between the start of mating and successful copulation)
- gestation index = number of females with liveborn pups*100/number of delivering pregnant females
- pre-implantation loss = number of corpora lutea- number of implantation sites*100/number of corpora lutea
- prenatal loss = number of implantation sites - number of pups delivered*100/number of implantation sites
- perinatal loss = number of stillborn pups*100/ number of pups delivered
- % liveborn pups (i.e. live birth index) = number of pups born alive*100/total number of pups delivered
- viability index day 4-21= number of pups surviving 21 days*100/number of liveborn after culling day 4
- sex ratio day n = number of live male pups on day n*100/ number of live pups on day n
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- F0-generation: During premating, several females started to develop sparsely haired areas and/or encrustations on the back from week 3 onwards (in all dose-groups). This was due to the ventilation holes in the exposure tubes. The incidence decreased after adapting the tubes by closing the holes. On 6 February 2012 and 7 February 2012, two females of the high-dose group were found dead (lactation days 16 and 17, respectively). At necropsy, no apparent cause of death could be found. Treatment-related clinical signs were not observed in the F0-generation, including the animals found dead. All signs observed were within the range of expected for animals of this strain and age.
- F1-generation: On 5 June 2012, one female of the high-dose group was found dead (lactation day 19). At necropsy, no apparent cause of death could be found. No treatment-related clinical signs were observed in the F1-generation during the study. All signs observed were within the range of expected for animals of this strain and age. Because several animals had not achieved sexual maturation minimally at PN 56 (females) or PN80 (males), vaginal patency and balanopreputial separation were registered as clinical signs from this period onwards.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Premating period:
- Males of the F0-generation: No statistically significant differences were observed on mean body weight during the study. Body weight change in low-dose males was statistically significantly decreased from day 49 to 56 and increased from day 56 to 63 during the study. This was considered as incidental finding, based on the absence of an effect on mean body weights and a dose-relation.
- Females of the F0-generation: No significant differences were observed on body weight or body weight change during premating.
- Males of the F1-generation: No statistically significant differences were observed on mean body weight during the study. From days 49 to 56 body weight change was statistically significantly decreased in high-dose males. A statistically significantly increased body weight change was observed in low-, mid-, and high dose males from day 56 to 63. These findings were not dose-related and are therefroe considered to be of no toxicologically significance.
- Females of the F1-generation: No significant differences were observed on body weight or body weight change during premating, except for an incidental statistically significant increase in body weight change in high-dose females from day 63 to 70 of the study.
Gestation period
- F0-generation: No statistically significant differences in mean body weights and body weight changes were observed between the exposed groups and the control group.
- F1-generation: No statistically significant difference in mean body weight was observed between the exposed groups and the control group. An incidental statistically significant increase in body weight change was observed in mid-dose females from day 7 to 14 only.
Lactation period:
- F0-generation: No statistically significant differences in mean body weights and body weight changes were observed between the exposed groups and the control group.
- F1-generation: No statistically significant differences in mean body weights and body weight changes were observed between the exposed groups and the control group.

FOOD CONSUMPTION (PARENTAL ANIMALS)
Premating period:
- Males and females of the F0-generation: No statistically significant differences on mean food consumption (expressed as g/animal/day and g/kg BW/day) were observed in males and females during the premating, except for an incidental increase in mean food consumption (g/kg BW/day) in males of the low-and mid-dose groups between days 42 and 49.
- Males and females of the F1-generation: No statistically significant differences on mean food consumption (expressed as g/animal/day and g/kg/day) were observed in males and females during the premating. Food consumption (expressed as g/kg BW/day) was statistically significantly increased in high-dose males from day 7 to 14 and in low-, mid, and high-dose males from day 56 to 63 of the study. These findings were considered to be incidental, because no relation with the concentration of the test substance was observed.
Gestation period:
- F0-generation: No statistically significant effect on mean food consumption (expressed as g/animal/day and g/kg BW/day) was observed during the gestation period.
- F1-generation: Food consumption (expressed as g/animal per day) was statistically significantly increased in mid-dose females from day 7 to 14 during gestation, which was considered to be incidental. No effect on mean food consumption (expressed as g/kg BW/day) was observed during the gestation period.
Lactation period:
- F0-generation: No statistically significant effect on mean food consumption (expressed as g/animal/day) was observed during the lactation period.
- F1-generation: No statistically significant effect on mean food consumption (expressed as g/animal/day) was observed during the lactation period.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
- F0-generation: A statistically significantly decreased mean cycle length was observed in females of the high-dose group. This was not considered as treatment-related, because a cycle length of 4-5 days is normal for this strain. In the control group more females with a cycle length of 6 or more days were observed compared to the high-dose group.
- F1-generation: No differences were observed in estrus cyclicity parameters.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Epididymal sperm motility
- F0-generation: No statistically significant differences were observed in sperm motility between the exposed groups and the control group.
- F1-generation: A statistically significantly decreased linearity (ratio VSL/VCL) was observed in the mid- and high-dose groups. Because none of the other sperm motility parameters were affected and the number of pregnant females and pups per litter were comparable between the groups, this finding was considered not to be treatment-related. Furthermore, histopathological examination of the epididymides and testes did not show treatment-related abnormalities.
Epididymal sperm count
- F0- and F1-generation: Epididymal sperm count in the exposed groups and the control group was comparable in both F0- and F1-genarations.
Sperm morphology
- F0- and F1-generation: No statistically significant differences were observed in sperm morphology between the high-dose group and the control group in both F0- and F1-generations.
Homogenisation resistant sperm
- F0- and F1-generation: Homogenisation resistant sperm count and daily sperm production in the testis were comparable between the high-dose group and the control group in both F0- and F1-generations.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Fertility:
- F0-generation: Each group comprised 28 males and females that were mated. Twenty seven, 26, 24 and 26 females of the control, low-, mid-, and high-dose group, respectively were found inseminated, of which 25, 21, 21, and 19, respectively were pregnant. The mean number of mating days until day 0 post coitum was comparable between all groups. No treatment-related differences were observed in the female and male mating indices. Both female and male fertility indices tended to be decreased in the high-dose group.
- F1-generation: Each group comprised 28 males and females that were mated. Twenty five, 28, 27 and 27 females of the control, low-, mid-, and high-dose group, respectively were found inseminated, of which 22, 23, 25, and 26, respectively were pregnant. No statistically significant differences were found in the mean number of mating days until day 0 post coitum, however, 5 of the animals of the control group were inseminated relatively late (between mating days 5 and 8). No treatment-related differences were observed in the female and male mating and fertility indices.
Delivery and litter data:
- F0-generation: Delivery Report: No treatment-related effects were observed on the duration of the gestation period and gestation index. All pregnant females delivered a litter. One, 1, 3 and 3 females of the control, low-, mid- and high-dose group delivered a litter with stillborn pups, of which one female of the mid-dose group and one female of the high-dose group delivered one stillborn pup only.
Litter Report: No treatment-related effects of the test substance were observed on the mean number of corpora lutea, implantation sites, preimplantation loss, mean number of pups delivered, prenatal and perinatal loss in the F0-generation. Five females were killed interim, because they had complete litter loss during the lactation period: Females 129 of the mid-dose group and female 203 of the highdose group both delivered 1 stillborn pup. Females 125, 153 and 159 of the middose group lost their complete litter due to cannibalism at PN 4, 7 and 4, respectively. None of these dams nor pups showed clinical signs or abnormal body weight.
- F1-generation: Delivery Report: The mean number of gestation days and gestation index were comparable between the exposed and control group. All pregnant females delivered a litter. One, 3,1 and 1 females of the control, low-, mid- and high-dose group delivered a litter with both live and stillborn pups.
Litter Report: No treatment-related effects of the test substance were observed on the mean number of corpora lutea, implantation sites, preimplantation loss, mean number of pups delivered, prenatal and perinatal loss of the F2 pups. One dam of the high-dose group (no. 715) was killed interim 24 May 2012, because she had complete litter loss during the lactation period (one pup delivered which was missing at day 1 of lactation). Pup 217-07 had been selected as female 715 for the F1-generation. This pup had a low pup weight when selected and body weight was low during premating and gestation. This dam had a low body weight at the start of lactation (144.7 gram; 32% decrease versus mean control body weights on day 0 of lactation) and her single pup was missing on day 1 of lactation. No histopathological abnormalities were observed in this female.
Cannibalism was mainly observed in the F0-generation. No relationship could be observed between cannibalism and decreased food intake of the dams or cage position in the animal room. The incidence of cannibalism as observed in the middose group of the F0-generation was within the range of historical control data of this strain and therefore not considered to be an adverse effect. The number of pups delivered and the number of stillborn pups were not considered to be adversely affected by test substance exposure.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- F0-generation: The following statistically significant differences were observed in organ weights: Relative liver weight was increased in males of the low-, mid-, and high-dose groups (about 5% compared to the control group). There was no dose-related response. Relative weight of the kidneys was increased in males of the mid-, and high-dose groups (about 5% compared to the control group). A clear dose-relation could not be observed and therefore this finding was considered to be not related to treatment. Absolute heart weight was increased in females of the high-dose group. Relative heart weight also tended to be increased in the high-dose group, although not statistically significant. Both absolute and relative weight of the adrenals was decreased in the females of the mid-dose-group. Due to the absence of this finding in the high-dose group, this was considered to be of no toxicological relevance.
- F1-generation: The following statistically significant differences were observed in organ weights: Relative weight of the adrenals was decreased in males of the mid-dose-group. Due to the absence of this finding in the high-dose group, this was considered to be not related to treatment. Both absolute and relative weight of the epididymis were decreased in low-dose males. This was an incidental finding, because no dose-response was observed. Relative liver weight was increased in females of the high-dose group (about 7% compared to the control group). Absolute and relative thyroid weights were decreased in females of the high dosegroup (about 8% compared to the control group).

GROSS PATHOLOGY (PARENTAL ANIMALS)
Intercurrent deaths
- F0-generation: Two female animals of the high-dose group (no. 177 and 189) were found dead on 6 February 2012 (lactation day 16) and 7 February 2012 (lactation day 17), respectively. At necropsy the lungs of animal 177 were red and did not collaps properly. No other macroscopic findings were found. Animal 189, showed red lungs, a swollen stomach and caecum, a red thymus and large adrenals.
- F1-generation: One female animal of the high-dose group (no. 677) was found dead on 5 June 2012 (lactation day 19). At necropsy, the stomach was swollen and its mucosa was brown. The jejunum was red. The caecum was swollen and the ovaries were red. The lungs showed red patches and were not collapsed properly. The uterus had a dark appearance.
Scheduled necropsies
- F0-generation: Macroscopic analysis revealed no treatment-related changes.
- F1-generation: Macroscopic analysis revealed no treatment-related changes.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- F0-generation: Microscopic analysis revealed no treatment-related changes. In agreement with the macroscopic findings, the lungs of animal 177 were hyperaemic. No obvious cause of death could be established. In agreement with the macroscopic findings, the lungs of animal 189 were hyperaemic. No abnormalities could be detected in the stomach. The caecum was necrotic. The thymus showed microhaemorrhages and the adrenals were hyperaemic. No obvious cause of death could be established.
- F1-generation: Microscopic analysis revealed no treatment-related changes. In agreement with the macroscopic findings, the lungs of animal 677 were hyperaemic. No abnormalities could be detected in the stomach or the jejunum. The caecum was necrotic. The ovaria were hyperaemic. Microscopic examination of the uterus revealed no explanation for the dark appearance. No obvious cause of death could be established.
Dose descriptor:
NOEL
Remarks:
Parental toxicity
Effect level:
5 000 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mortality of two F0-females in the high dose group.
Dose descriptor:
NOEL
Remarks:
Fertility
Effect level:
15 000 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on fertility parameters were observed.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
VIABILITY (OFFSPRING)

CLINICAL SIGNS (OFFSPRING)
- F0-generation: The number of runts was comparable between the exposed groups and the control group. Treatment-related pup clinical signs were not observed. All signs observed were within the range of the expected for this strain and age. The right eye of pup 123-05 had encrustations at PN14 and was missing at PN21. Because this pup was selected for the F1-generation as female 655 this clinical sign persisted. A high incidence of early pup mortality (PN0-4) in the mid-dose group was observed. This was due to a high incidence of cannibalism and not considered an adverse effect. The pups of dams 177 and 189 were killed interim, because the dams were found dead. The mean number of live pups was comparable in the exposed groups compared to the control group at PN4, 7, 14 and 20. Viability index from PN4-21 and sex ratio were comparable between the groups.
- F1-generation: A statistically significant number of runts was observed in female pups of the middose group on PN4 and PN7. This finding was not dose-related and therefore not considered as adverse effect. Treatment-related pup clinical signs were not observed. All signs observed were within the range of the expected for this strain and age. There was no treatment related-effect on the number of dead pups between PN0-4. The mean number of live pups was comparable in the exposed groups compared to the control group at PN4, 7, 14 and 20. Viability index from PN4-21 and sex ratio were comparable between the groups.

BODY WEIGHT (OFFSPRING)
- F0-generation: No statistically significant differences in mean pup weights and pup weight changes were observed between the pups of the exposed groups and the control group.
- F1-generation: Statistically significantly increased mean pup body weights were observed only in females of the high-dose group at lactation days 0, 4, and 7. Mean pup body weight was statistically significantly increased when combining male and females pup weights in the high-dose group at day 4 and 7. No statistically significant differences in pup weight changes were observed between the pups of the exposed groups and the control group.

SEXUAL MATURATION (OFFSPRING)
In the F1-male and female animals, no statistically significant difference was observed between the day of achievement of balanopreputial separation or vaginal patency between the exposed groups and the control group.

ORGAN WEIGHTS (OFFSPRING)
- F0-generation: No statistically significant differences in absolute and relative mean pup organ weights and pup weight changes were observed between the pups of the exposed groups and the control group.
- F1-generation: No statistically significant differences in absolute and relative mean pup organ weights and pup weight changes were observed between the pups of the exposed groups and the control group.

GROSS PATHOLOGY (OFFSPRING)
- F0- and F1generation: No abnormalities were observed

HISTOPATHOLOGY (OFFSPRING)
- F0- and F1generation: No abnormalities were observed
Dose descriptor:
NOEL
Remarks:
Parental toxicity
Generation:
F1
Effect level:
5 000 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Mortality of one F1-female of the high-dose group.
Dose descriptor:
NOEL
Remarks:
Fertility
Generation:
F1
Effect level:
15 000 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on fertility parameters were observed.
Dose descriptor:
NOEL
Remarks:
Developmental
Generation:
F1
Effect level:
15 000 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on offspring were observed.
Dose descriptor:
NOEL
Remarks:
Developmental
Generation:
F2
Effect level:
15 000 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects on offspring were observed.
Reproductive effects observed:
not specified
Conclusions:
Exposure of rats to 2,000, 5,000 or 15,000 ppm of the test substance for 6 h/day, 5 days a week during premating and daily during mating, gestation up to GD19 and lactation (from PN5 onwards) resulted in mortality of two F0-females and one F1-female of the high-dose group at the end of the lactation period. Based on these results, the No Observed Effect Level (NOEL) for parental toxicity for exposure to the test substance by inhalation, under the conditions of this two-generation reproduction toxicity study in rats, is considered to be 5,000 ppm (26550 mg/m3). The NOEL for fertility and development for exposure to the test substance by inhalation, under the conditions of this two-generation reproduction toxicity study in rats, is considered to be 15,000 ppm (79650 mg/m3), because no adverse effects on fertility parameters or offspring were observed.
Executive summary:

In this two-generation study, performed in accordance with OECD Guideline 416 and GLP, 28 Wistar rats/sex/group were exposed by inhalation to 0 (air), 2,000, 5,000 or 15,000 ppm of the test substance. Male animals were exposed to the test substance in air (nose-only) for at least 10 weeks prior to mating during 6 hours/day and 5 days/week, and daily during mating for 6 hours/day. Males were exposed until sacrifice after at least 11 weeks (for sperm analyses and necropsy). The females were exposed to the test substance in air (nose-only) for at least 10 weeks prior to mating for 6 hours/day and 5 days/week, and daily during mating and up to gestation day (GD) 19 for 6 hours/day. From day 5 of lactation onwards, females were exposed daily by whole body exposure for 6 hours/day to the test substance until the end of the lactation period (PN21) or the sacrifice of the females shortly thereafter. From PN day 22 up to ca. 6 weeks of age, the F1-generation male and female pups were exposed by whole body exposure during 6 hours/day and 5 days/week. Subsequently, F1-generation male animals were exposed by nose-only exposure until the end of the premating period during 6 hours/day and 5 days/week, and daily for 6 hours/day during mating and up to sacrifice after at least 11 weeks (for sperm analyses and necropsy). F1-generation female animals were exposed by nose-only exposure until the end of the premating period during 6 hours/day and 5 days/week, and daily during mating and up to GD19 for 6 hours/day. From PN5 onwards, F1-generation females were exposed daily 6 hours/day to the test substance by whole body exposure until sacrifice on or shortly after PN21. Clinical signs did not reveal treatment-related changes. Two females of the high-dose group in the F0-generation and one female of the high-dose group in the F1 -generation were found dead on lactation days 16, 17 and 19. Histopathological observation of these animals revealed no obvious cause of death. Due to the incidence of mortality in the high-dose group only, this was considered to be related to exposure to the test substance. No treatment-related effects were observed on body weights, body weight changes and food consumption. No adverse effects of the test substance were observed on estrus cycle parameters of the F0- and F1-female animals and on sperm parameters of the male F0- and F1 -animals. The number of pregnant F0-females was 25, 21, 21, and 19 in the control, low-,mid- and high-dose group, respectively. The number of pregnant F1-females was 22, 23, 25 and 26 in the control, low-, mid- and high-dose group, respectively. Precoital time, gestation index and duration of gestation, male and female fertility indices, incidences of dams with live- and stillborn pups were not affected by exposure to the test substance by inhalation in either generation. In both generations, the mean number of pups delivered, the incidences of liveborn and stillborn pups, the number of live and dead pups at delivery, the number of pups lost during the lactation period, the sex ratio, pup clinical observations, pup organ weight and macroscopic observations were considered not to be affected by exposure to the test substance. The increased pup body weights observed in F1-female pups on PN day 0, 4 and 7 were considered not to be an adverse effect of the test substance, based on the absence of a clear dose-reponse and reduction of the effect on PN14 and 21. No statistically significant differences were observed among the various groups in timing of balanopreputial separation or vaginal patency. Organ weights of parental animals of both generations were considered not to be affected by exposure to the test substance. The slight significant differences observed in liver, kidney and thyroid weight were not consistently observed in both generations, not dose-related and not accompanied by corroborative histopathological effects. Therefore no toxicological significance is attached to these slight organ weight changes. At necropsy of the parental animals no test substance related gross changes were observed in the F0- and F1-generation animals. Microscopic examination did not reveal any test substance related findings in the male and female animals of the F0- and F1-generations. In conclusion, exposure of rats to 2,000, 5,000 or 15,000 ppm to the test substance for 6 h/day, 5 days a week during premating and daily during mating, gestation up to GD19 and lactation (from PN5 onwards) resulted in mortality of two F0-females and one F1-female of the high-dose group at the end of the lactation period. Based on these results, the No Observed Effect Level (NOEL) for parental toxicity for exposure to the test substance by inhalation, under the conditions of this two-generation reproduction toxicity study in rats, is considered to be 5,000 ppm (26550 mg/m3). The NOEL for fertility and development for exposure to the test substance by inhalation, under the conditions of this two-generation reproduction toxicity study in rats, is considered to be 15,000 ppm (79650 mg/m3), because no adverse effects on fertility parameters or offspring were observed.

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Species:
rat
Quality of whole database:
OECD guideline 416 2-generation study is available, performed in compliance with GLP. This study is sufficiently adequate for this endpoint, well conducted and documented.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In this two-generation study, performed in accordance with OECD Guideline 416 and GLP, 28 Wistar rats/sex/group were exposed by inhalation to 0 (air), 2,000, 5,000 or 15,000 ppm of the test substance. (TNO Triskelion 2012) Male animals were exposed to the test substance in air (nose-only) for at least 10 weeks prior to mating during 6 hours/day and 5 days/week, and daily during mating for 6 hours/day. Males were exposed until sacrifice after at least 11 weeks (for sperm analyses and necropsy). The females were exposed to the test substance in air (nose-only) for at least 10 weeks prior to mating for 6 hours/day and 5 days/week, and daily during mating and up to gestation day (GD) 19 for 6 hours/day. From day 5 of lactation onwards, females were exposed daily by whole body exposure for 6 hours/day to the test substance until the end of the lactation period (PN21) or the sacrifice of the females shortly thereafter. From PN day 22 up to ca. 6 weeks of age, the F1-generation male and female pups were exposed by whole body exposure during 6 hours/day and 5 days/week. Subsequently, F1-generation male animals were exposed by nose-only exposure until the end of the premating period during 6 hours/day and 5 days/week, and daily for 6 hours/day during mating and up to sacrifice after at least 11 weeks (for sperm analyses and necropsy). F1-generation female animals were exposed by nose-only exposure until the end of the premating period during 6 hours/day and 5 days/week, and daily during mating and up to GD19 for 6 hours/day. From PN5 onwards, F1-generation females were exposed daily 6 hours/day to the test substance by whole body exposure until sacrifice on or shortly after PN21. Clinical signs did not reveal treatment-related changes. Two females of the high-dose group in the F0-generation and one female of the high-dose group in the F1 -generation were found dead on lactation days 16, 17 and 19. Histopathological observation of these animals revealed no obvious cause of death. Due to the incidence of mortality in the high-dose group only, this was considered to be related to exposure to the test substance. No treatment-related effects were observed on body weights, body weight changes and food consumption. No adverse effects of the test substance were observed on estrus cycle parameters of the F0- and F1-female animals and on sperm parameters of the male F0- and F1 -animals. The number of pregnant F0-females was 25, 21, 21, and 19 in the control, low-,mid- and high-dose group, respectively. The number of pregnant F1-females was 22, 23, 25 and 26 in the control, low-, mid- and high-dose group, respectively. Precoital time, gestation index and duration of gestation, male and female fertility indices, incidences of dams with live- and stillborn pups were not affected by exposure to the test substance by inhalation in either generation. In both generations, the mean number of pups delivered, the incidences of liveborn and stillborn pups, the number of live and dead pups at delivery, the number of pups lost during the lactation period, the sex ratio, pup clinical observations, pup organ weight and macroscopic observations were considered not to be affected by exposure to the test substance. The increased pup body weights observed in F1-female pups on PN day 0, 4 and 7 were considered not to be an adverse effect of the test substance, based on the absence of a clear dose-reponse and reduction of the effect on PN14 and 21. No statistically significant differences were observed among the various groups in timing of balanopreputial separation or vaginal patency. Organ weights of parental animals of both generations were considered not to be affected by exposure to the test substance. The slight significant differences observed in liver, kidney and thyroid weight were not consistently observed in both generations, not dose-related and not accompanied by corroborative histopathological effects. Therefore no toxicological significance is attached to these slight organ weight changes. At necropsy of the parental animals no test substance related gross changes were observed in the F0- and F1-generation animals. Microscopic examination did not reveal any test substance related findings in the male and female animals of the F0- and F1-generations. In conclusion, exposure of rats to 2,000, 5,000 or 15,000 ppm to the test substance for 6 h/day, 5 days a week during premating and daily during mating, gestation up to GD19 and lactation (from PN5 onwards) resulted in mortality of two F0-females and one F1-female of the high-dose group at the end of the lactation period. Based on these results, the No Observed Effect Level (NOEL) for parental toxicity for exposure to the test substance by inhalation, under the conditions of this two-generation reproduction toxicity study in rats, is considered to be 5,000 ppm (26550 mg/m3). The NOEL for fertility and development for exposure to the test substance by inhalation, under the conditions of this two-generation reproduction toxicity study in rats, is considered to be 15,000 ppm (79650 mg/m3), because no adverse effects on fertility parameters or offspring were observed.


Short description of key information:
Exposure of rats to 2,000, 5,000 or 15,000 ppm of the test substance for 6 h/day, 5 days a week during premating and daily during mating, gestation up to GD19 and lactation (from PN5 onwards) resulted in mortality of two F0-females and one F1-female of the high-dose group at the end of the lactation period. Based on these results, the No Observed Effect Level (NOEL) for parental toxicity for exposure to the test substance by inhalation, under the conditions of this two-generation reproduction toxicity study in rats, is considered to be 5,000 ppm (26550 mg/m3). The NOEL for fertility and development for exposure to the test substance by inhalation, under the conditions of this two-generation reproduction toxicity study in rats, is considered to be 15,000 ppm (79650 mg/m3), because no adverse effects on fertility parameters or offspring were observed.

Justification for selection of Effect on fertility via inhalation route:
One OECD guideline 416 2-generation study, performed in compliance with GLP, is available. This study is sufficiently adequate for this endpoint.

Effects on developmental toxicity

Description of key information
The inhalation developmental toxicity study (OECD 414, GLP) in rabbits, showed no adverse effects on development up to the highest dose tested. Therefore, the NOAEL for maternal and developmental toxicity in rabbits was determined to be 15000 ppm (79650 mg/m3). In the inhalation developmental toxicity study (OECD 414, GLP) in rats an increased incidence of dilated urinary bladders in fetuses of the high-dose group (15000 ppm) was considered to be a treatment-related effect. Therefore, the NOAEL for developmental toxicity in rats was determined to be 10000 ppm (53100 mg/m3). The significance of the increased incidence of dilated urinary bladders in fetuses at 15000 ppm is not clear because no other possibly related effects, such as kidney abnormalities or increased amnion fluid, were observed. As, moreover, no effects on the offspring were observed in the 2-generation study, this finding is considered not to warrant classification. Maternal toxicity was not observed up to and including the highest dose tested. Therefore, the NOAEL for maternal toxicity in rats was determined to be 15000 ppm.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to generally valid procedures and according to GLP guidelines. All parameters described are closely related or comparable to testing guideline methods.
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Triskelion, Utrechtseweg 48, 3404 HE Zeist, the Netherlands
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charlese River Deutschland, Sulzfeld, Germany
- Age at study initiation: females were 11 weeks old at mating; males were 13 weeks old at mating
- Weight at study initiation: Female weights ranged from 196.90 to 201.16 g on gestation day 0.
- Fasting period before study: None
- Housing: The rats were housed in macrolon cages with a bedding of wood shavings and strips of paper (Enviro-dri) as environmental enrichment. During the quarantine and acclimatisation period, the males were housed individually and the females were housed in groups of 4. For mating, one male and two females were housed together in type 3 macrolon cages. Mated females were housed individually in macrolon cages which were placed in a separate cage rack. The location of the mated females in the new cage racks were determined by the date of mating (females found sperm-positive on the same date were considered a "lot") and by the animal number (within each lot the mated females was housed in the order of animal number).
During exposure periods, the rats were individually housed in the exposure unit.
- Diet: The rats received a cereal-based (closed formula) rodent diet (Rat & Mouse No. 3 Breeding Diet, RM3) from a commercial supplier (SDS Special Diets Services, Witham, England). Feed was provided ad libitum from the arrival of the rats until the end of the study, except during exposure.
- Water: The drinking water (tap-water) was given in polypropylene bottles, which were cleaned weekly and filled as needed. Drinking water was provided ad libitum from the arrival of the rats until the end of the study, except during exposure.
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Details on exposure:
EEPOSURE EQUIPMENT:
Animals were exposed to the test atmosphere in nose-only exposure units. Each unit consisted of a cylindrical PVC column with a volume of ca. 70 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column, and was exhausted at the top. Each column included three rodent tube sections and each rodent tube section accommodated 20 ports for animal exposure. Additional ports were used for test atmosphere sampling, measurement of oxygen concentration, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column (males and females alternated). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. The location of the rats was changed weekly by rotating the animals 5 places clockwise and moving the animals from the upper section to the middle section, the animals from the middle section to the lower section and the animals from the lower section to the upper section. The units were illuminated externally by normal laboratory TL-lighting.

GENERATION OF TEST ATMOSPHERE:
- The inhalation equipment was designed to expose the rats to a continuous supply of fresh test atmosphere. To generate the test atmosphere, a flow of cooled liquid test material controlled by a peristaltic pump was allowed to evaporate in a flow of humidified air (mass flow controlled). The air was supplemented with mass flow controlled oxygen for the mid and high concentrations to ensure a sufficiently high and equal oxygen concentration. Total test atmosphere flow was between 54 and 55 L/min for all groups except on one day, where it was between 29 and 30 L/min. At all times the flow was more than 1 L/min/animal with regard to the amount of animals present. The exposure unit for control animals was supplied with a mass flow controlled stream of humidified compressed air only.
- The measured concentrations were used in a PI feedback system to control the peristaltic pumps. The feed back system took into account the proportional (P) and the integrated deviations (I) of the concentrations from the set point.
- The settings of the mass flow controllers were checked each morning at the start of the generation and subsequently at regular intervals during exposure (approximately bi-hourly, i.e. three times a day).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ACTUAL CONCENTRATIONS:
- The actual concentration of the test material in the atmospheres was measured by total carbon analysis. The test atmospheres were sampled from the exposure units at the animals’ breathing zone and were passed to total carbon analyzers. The response of the analyzers was recorded on a pc every minute using a CAN transmitter.
- The daily mean response was calculated by averaging values read every minute.
- The total carbon analyzers were calibrated by sampling from 3 concentrations (in duplicate) in a range including the target concentration. The concentrations of the test material used to calibrate the total carbon analyzer were prepared in a sample bag by injecting known amounts (by mass) of the (cooled) test material.
Details on mating procedure:
At the start of mating, 2 nulliparous females were caged with each male for mating until a sperm positive vaginal smear was detected. Every consecutive morning vaginal examinations were made to ascertain copulation by detection of sperm cells in a vaginal smear. Upon evidence of copulation, positive females were housed individually. The day a sperm-positive smear is detected was considered as gestation day 0. The mated females were distributed over the 4 experimental groups in such a way that the animals from the same day of pregnancy were, as far as possible, equally distributed over all groups. Females mated by the same male were placed in different groups.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
From gestation day 6 up to and including gestation day 19.
Duration of test:
In-life termination of female rats occurred on gestation day 21.
Remarks:
Doses / Concentrations:
0, 4000, 10000 and 15000 ppm
Basis:
other: Target concentrations
Remarks:
Doses / Concentrations:
0, 3988, 9981 and 14906 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
24 pregnant female rats
Control animals:
yes
Details on study design:
- Dose selection rationale: The concentration levels were selected in consultation with the sponsor and were the same as used in the sub-chronic (90-day) inhalation toxicity study in rats (TNO Triskelion study V8964).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily and, if necessary, the animal was handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon, especially for dead or moribund animals. In weekends and on public holidays only one check per day was carried out. All abnormalities, signs of ill health, reaction to treatment and mortality were recorded.

CLINICAL OBSERVATIONS DURING EXPOSURE:
- Time schedule: A group-wise observation was made halfway through each exposure day.

BODY WEIGHT: Yes
- Time schedule for examinations: On gestation days 0, 3, 6, 9, 12, 15, 19 and 21.

FOOD CONSUMPTION: Yes
- The food consumed for each mated female was measured over the periods: gestation days 0-3, 3-6, 6-9, 9-12, 12-15, 15-19 and 19-21.
- The results were expressed in g per animal per day and g per kg body weight per day.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: The uteri (including the fetuses), ovaries and placentas of all females.
Ovaries and uterine content:
The uteri (including the fetuses), ovaries and placentas of all females killed on gestation day 21 were examined for the following parameters: number of corpora lutea, number of implantation sites, number of early and late resorptions, number of live and dead fetuses, sex of the fetuses, number of grossly visible malformed fetuses and fetuses with external abnormalities, weight of ovaries, weight of uterus, containing placentas and fetuses, weight of empty uterus, weight of live fetuses, weight of the placentas of live fetuses, gross evaluation of placentas.
Fetal examinations:
- External examinations: Yes: [all]
- Soft tissue examinations: Yes [half per litter ]
- Skeletal examinations: Yes [half per litter ]
Statistics:
The resulting data were analyzed using the methods mentioned below. As a level of significance was considered: p< 0.05.
- Clinical findings were evaluated by Fisher's exact probability test.
- Body weight, body weight gain, organ weights and food consumption data were subjected to one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison tests.
- Fisher's exact probability test was used to evaluate the number of mated and pregnant females and females with live fetuses.
- Number of corpora lutea, implantation sites, live and dead fetuses and early and late resorptions were evaluated by Kruskal-Wallis nonparametric analysis of variance followed by the Mann Whitney U-test.
- Mortality data, data of the pathology of parent females and the fetopathological screening were evaluated by the Fisher’s exact probability test.
Indices:
As far as applicable for each group the following indices were recorded:
- female fecundity index = (number of pregnant females/number of females mated) x 100
- pre-implantation loss = [(number of corpora lutea - number of implantation sites)/ number of corpora lutea] x 100
- post-implantation loss = [(number of implantation sites- number of live fetuses)/number of implantation sites] x 100
- gestation index = (number of females with live fetuses/number of females pregnant) x 100
- sex ratio = (number of live male fetuses/number of live fetuses) x 100
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- Clinical signs and mortalities: Daily clinical observations during gestation did not reveal any remarkable findings in animals’ appearance, general condition or behaviour among the test and control groups. None of the animals died during the study.

- Body weight and body weight change: No statistically significant differences in body weight and body weight change were observed between the females of the test groups and the control group.

- Food consumption: Food consumption (both g/kg body weight/day and g/animal/day) of the test groups did not differ from the control group.

- Reproduction and litter data: In each group, 24 females were mated. A total of 18, 17, 20 and 18 females of the control, low-, mid-, and high-concentration test substance groups, respectively, appeared to be pregnant. One female of the control group had no viable fetuses including a late resorption. No statistically significant differences were observed in the gestation index and female fecundity index among the groups. No statistically significant differences were observed in the number of corpora lutea, implantation sites, pre- and post-implantation loss, live and dead fetuses, resorptions and sex ratio between test groups and control group.

- Macroscopic findings in dams at necropsy: No statistically significant differences were observed in the incidence of parental necropsy observations among the groups. The findings observed were incidental and not related to treatment.

- Organ weights and net weight change: The mean weight of the gravid uterus, carcass, net weight change from day 0, empty uterus and ovaries of the groups exposed to the test substance did not differ from the control group.
Dose descriptor:
NOEL
Effect level:
15 000 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEL
Effect level:
10 000 ppm
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes. Remark: A statistically significant increase was observed in the incidence of a dilated urinary bladder in fetuses of the high-concentration (15,000 ppm) group.

Details on embryotoxic / teratogenic effects:
- Fetal external observations: There were no treatment-related statistically significant differences in fetal external observations. One female of the low-concentration test substance group showed a fetus with a soft skull, malformed ears and subcutaneous oedema. Because no similar effect was observed in the mid- and high-dose groups, these findings were considered as incidental and not related to treatment. At skeletal examination of the one female fetus of the low-concentration test substance group, only incomplete ossification of the frontal, parietal, interparietal and supra occipital bones was observed. Other fetal external observations that occurred at low incidences in all groups included small fetuses and small subcutaneous haemorrhages/petechiae on the skin. These incidental observations are common for this strain and not related to treatment.

- Findings of the placenta: In one female of the control group, a large placenta and an amnion sac filled with red fluid were observed with a late resorption. This female had no viable fetuses. No treatment-related findings of the placenta were observed.

- Fetal and placental weight: No statistically significant differences were observed on placenta and fetal weight among the groups.

- Visceral malformations: No visceral malformations were observed.

- Visceral anomalies: A statistically significant increase was observed in the incidence of a dilated urinary bladder in fetuses of the high-concentration test substance group. The significance of this finding is not clear, as no other possibly related effects, such as kidney abnormalities or increased amnion fluid were observed in this study. However, this visceral anomaly occurred more frequently with increasing doses of the test substance. Therefore this finding is considered to be a treatment-related effect. One fetus of the mid-concentration group showed diverticulum of the intestines. This finding occurred in the mid-concentration group only; therefore, this finding was considered not to be a treatment-related effect. No other statistically significant effects in the incidence of visceral anomalies were observed among the groups.

- Visceral variations: Visceral variations in all groups included haemorrhagic areas in the oral cavity, haemorrhagic areas in the nasal cavity, folded retinas, not well-defined soft lenses, pericard and stomach filled with haemorrhagic fluid, and kinked and bent ureters. The incidences of folded retina and stomach containing haemorrhagic fluid were statistically significantly decreased in the high-concentration test substance group and the low-concentration group, respectively. Overall, the total incidence of fetal visceral variations was decreased in the low- and high-concentration groups compared to the control group.

- Skeletal malformations: No skeletal malformations were observed.

- Skeletal anomalies: No skeletal anomalies were observed.

- Skeletal variations: Skeletal variations were seen in parietal and interparietal bones (supernumerary), supraoccipital bones (holes), ribs (accessory lumbar ribs), and sternebrae (irregular ossification of one or irregular shape of one or more) and caudal bodies (one or two supernumerary). No statistically significant differences were observed in skeletal variations among the groups.

- Skeletal retardations: A few statistically significant differences were observed in the incidences of fetuses and/or litters showing skeletal retardations after treatment with the test substance: (1) Decreased fetal incidence of three or more incompletely ossified caudal arches in the low- and mid-concentration groups. (2) Decreased fetal incidence of 1-2 incompletely ossified metacarpals in the low-concentration group. The observed statistically significant differences in retardations of fetal skeletons were incidental, inconsistent and/or not dose-related. This kind of variation in skeletal ossification is considered as normal developmental variability. No other indications of developmental toxicity were observed. Therefore, these variations are considered to be not treatment-related.
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In conclusion, the increased incidence of dilated urinary bladders in fetuses of the high-dose group (15000 ppm or 79650 mg/m3) was considered to be a treatment-related effect. Therefore, the NOEL for developmental toxicity was determined to be 10000 ppm (53100 mg/m3). Maternal toxicity was not observed up to and including the highest dose tested. Therefore, the NOEL for maternal toxicity was determined to be 15000 ppm.
Executive summary:

In a developmental toxicity study performed according to OECD 414 and GLP, 24 pregnant Wistar rats per dose group were exposed by nose-only inhalation from Gestation Day (GD) 6 to GD 19. The animals were exposed every day to clean air (air control) or to the test substance at exposure concentrations of 4,000, 10,000 or 15,000 ppm for 6 hours per day. Inhalation of the test substance did not induce any relevant changes in the condition or macroscopic findings of the dams. No treatment-related effects were observed on body weight, body weight gain and food consumption of the females exposed to the test substance. Gestation index, female fecundity index, the number of corpora lutea, implantation sites, pre- and post implantation loss, live and dead fetuses, resorptions and sex ratio after exposure to the test substance did not differ from none-treated controls. Parental necropsy did not reveal any differences in reproductive organ weights and macroscopic findings. Fetal external- and placental observations and weights did not reveal any treatment-related effects. A statistically significant increase in the incidence of dilated urinary bladders was observed in fetuses of the high-dose group. The significance of this finding is not clear, as no other possibly related effects, such as kidney abnormalities or increased amnion fluid were observed in this study. However, this visceral anomaly occurred more frequently with increasing doses. Therefore this finding is considered to be a treatment-related effect. No treatment-related effects were observed on visceral malformations and variations, and on skeletal malformations, anomalies, variations, and retardations. In conclusion, the increased incidence of dilated urinary bladders in fetuses of the high-dose group was considered to be a treatment-related effect. Therefore, under the conditions of this study, inhalation of the test substance at concentrations up to 10,000 ppm during gestation of females was tolerated without obvious signs of developmental toxicity. Under the conditions of this study, inhalation of the test substance at concentrations up to 15,000 ppm during gestation of females was tolerated without obvious signs of maternal toxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
53 100 mg/m³
Species:
rat
Quality of whole database:
Two OECD guideline 414 developmental toxicity studies, one in rat and one in rabbit, performed in compliance with GLP, are available. Furthermore, one preliminary developmental toxicity study in rabbits is available. These studies are sufficiently adequate for this endpoint, well conducted and documented.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Rabbit

In a preliminary developmental toxicity study, based on OECD 414 (non-GLP), 8 female New-Zealand White rabbits per dose group were exposed by whole-body inhalation from Gestation Day (GD) 6 to GD 28, inclusively(GD 0 = day of mating confirmation) (Huntingdon 2010b). The animals were exposed every day to clean air (air control) or to the test substance at exposure concentrations of 2,500, 10,000 or 15,000 ppm for 6 hours per day. There were no effects of exposures on survival, clinical signs, body weights, food consumption or maternal or pup findings at sacrifice. Fetal visceral and skeletal examinations were not conducted. The NOAEL for adult pregnant female rabbits for systemic and reproductive and developmental toxicity was 15,000 ppm.

In a developmental toxicity study performed according to OECD 414 and GLP, 22 female New-Zealand White rabbits per dose group were exposed by whole-body inhalation from Gestation Day (GD) 6 to GD 28, inclusively(GD 0 = day of mating confirmation) (Huntingdon, 2010a). The animals were exposed every day to clean air (air control) or to the test substance at exposure concentrations of 2,500, 10,000 or 15,000 ppm for 6 hours per day. There were no effects of exposures on survival, clinical signs, body weights, food consumption, maternal or pup findings at sacrifice, or fetal external, visceral or skeletal findings. Therefore, the NOAEL for adult pregnant female rabbits for systemic and reproductive and developmental toxicity was 15,000 ppm.

Rat

In a developmental toxicity study performed according to OECD 414 and GLP, 24 pregnant Wistar rats per dose group were exposed by nose-only inhalation from Gestation Day (GD) 6 to GD 19 (TNO Triskelion, 2011b). The animals were exposed every day to clean air (air control) or to the test substance at exposure concentrations of 4,000, 10,000 or 15,000 ppm for 6 hours per day. Inhalation of the test substance did not induce any relevant changes in the condition or macroscopic findings of the dams. No treatment-related effects were observed on body weight, body weight gain and food consumption of the females exposed to the test substance. Gestation index, female fecundity index, the number of corpora lutea, implantation sites, pre- and post implantation loss, live and dead fetuses, resorptions and sex ratio after exposure to the test substance did not differ from none-treated controls. Parental necropsy did not reveal any differences in reproductive organ weights and macroscopic findings. Fetal external- and placental observations and weights did not reveal any treatment-related effects. A statistically significant increase in the incidence of dilated urinary bladders was observed in fetuses of the high-dose group. The significance of this finding is not clear, as no other possibly related effects, such as kidney abnormalities or increased amnion fluid were observed in this study. However, this visceral anomaly occurred more frequently with increasing doses. Therefore this finding is considered to be a treatment-related effect. No treatment-related effects were observed on visceral malformations and variations, and on skeletal malformations, anomalies, variations, and retardations. In conclusion, the increased incidence of dilated urinary bladders in fetuses of the high-dose group was considered to be a treatment-related effect. Therefore, under the conditions of this study, inhalation of the test substance at concentrations up to 10,000 ppm during gestation of females was tolerated without obvious signs of developmental toxicity. Under the conditions of this study, inhalation of the test substance at concentrations up to 15,000 ppm during gestation of females was tolerated without obvious signs of maternal toxicity.


Justification for selection of Effect on developmental toxicity: via inhalation route:
Two OECD guideline 414 developmental toxicity studies, one in rat and one in rabbit, performed in compliance with GLP, are available. These studies are sufficiently adequate for this endpoint.

Justification for classification or non-classification

In the two-generation study in rats no adverse effects on fertility parameters or offspring were observed. In the developmental toxicity study in rabbits no adverse effects on development were observed. In the developmental toxicity study in rats, a statistically significant increase in the incidence of dilated urinary bladders was observed in fetuses of the high-dose group. However, the significance of this finding is not clear, as no other possibly related effects, such as kidney abnormalities or increased amnion fluid were observed in this study. Moreover, in the 2 -generation study no effects on the offspring were observed. Therefore, this finding is not considered to warrant classification. In conclusion, classification is not necessary for toxicity to reproduction in accordance with EU Directive 67/548/EEC (DSD) and EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.