Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April - August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
An inhalation developmental toxicity with nitromethane in rats has been conducted. This study is used as read-acrcoss in this dossier. A document justifying the category/read-across approach has been attached.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
The overall purity of the lot of material used at the beginning of the test was approximately 98%. Gas chromatographic analyses showed that this lot contained

Test animals

Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
The study was conducted with albino rats. The rat was used because this species is considered suitable for this type of study, and is usually required by regulatory agencies. Male and female Wistar Han IGS rats (Crl:WI(Han)) were obtained from a colony maintained under SPF-conditions at Charles River Deutschland, Sulzfeld, Germany. This rat strain was used because it is routinely used at the test facility for this type of studies. In the study the female rats were about 10 weeks old and the male rats about 11 weeks old at arrival. The age difference between males and females was deliberate, to avoid mating of siblings. Male animals were used for mating only and were not part of the study otherwise. Upon arrival, the animals were housed in quarantine and checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status were carried out in a few randomly chosen rats of the lot delivered. Upon satisfactory serology results, the animals were transferred to their definitive animal room (or the quarantine room will be cleared for use as experimental room). The rats were acclimatized to the laboratory conditions for at least 5 days prior to the start of the exposure.

The animals used for the study were housed under conventional conditions in one animal room. The room was ventilated with about 10 air changes per hour and was maintained at a temperature of 20-24oC and a relative humidity of at least 45-65%. Lighting was artificial with a sequence of 12 hours light and 12 hours dark.

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The inhalation equipment was designed to expose the animals to a continuous supply of fresh test atmosphere. To generate the test atmospheres, a liquid flow of test material, controlled by a peristaltic pump (Watson-Marlow type 502s, Bredel Pumps Limited, Falmouth, Cornwall, England) was allowed to evaporate in a mass flow controlled (Bronkhorst Hi Tec, Ruurlo, the Netherlands) stream of dry compressed air, by directing it through a glass evaporator which was kept at a constant temperature of 37˚C by circulating heated water. For safety reasons, this was performed in a safety cabinet. The resulting test atmosphere was mixed with the main air stream (available as a laboratory provided source of non-pressurized, HEPA-filtered air), which was subsequently directed to the inlets at the top of the exposure chamber; the atmosphere was exhausted at the bottom. The exposure unit for the control animals was supplied with a controlled flow of HEPA-filtered air only.

The flow of the test atmosphere was controlled using a constant volume controller and was measured in the exhaust of the exposure chamber using a KIMO air velocity sensor (type CTV110-AOD150; KIMO, Emerainville, France). The air flow was continuously measured and recorded on a PC every minute, using a CAN transmitter (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The animals were placed in the exposure chamber prior to the start of the test atmosphere generation. Test atmosphere generation was stopped six hours after the start of generation. The animals were removed from the exposure chamber after the concentration had dropped below a level of 1% of the target concentration.

Prior to the first exposure of the animals, homogeneous distribution of the test material in the exposure chambers was confirmed by analysis of samples taken at five different locations in each exposure chamber (deviation of individual values from the mean of all five samples should not exceed 10%; the actual maximum deviation was 4.8% for the low-concentration group).

TEST ATMOSPHERE
The actual concentration of the test material in the test atmospheres was measured by total carbon analysis (Sick Maihak GMS 810 EuroFID Total Hydrocarbon Analyzer; Sick Instruments Benelux, Hedel, the Netherlands). 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 at one minute intervals 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. The average concentration during exposure was corrected for the duration of the animals’ stay in the exposure chamber and the number of measurements during exposure.

TOTAL AIR FLOW, TEMPERATTURE, RELATIVE HUMIDITY, OXYGEN AND CARBON DIOXIDE CONCENTRATION
The total flow of test atmosphere during exposure was continuously measured using air velocity sensors (type CTV110-AOD150; KIMO, Emerainville, France) in the chamber exhaust. The measurements of the air velocity sensors were recorded on a PC every minute using CAN transmitters (G. Lufft Mess- und Regeltechnik GmbH, 70719 Felbach, Germany). The responses of the sensors were converted to air flows by means of calibration graphs. Prior to the start of the exposure, the air velocity sensors were calibrated by measuring the response at a range of air flows (encompassing the flows used during the study) generated using mass flow controlled streams of clean dry air.

Temperature and relative humidity of the test atmospheres were measured continuously and recorded every minuteat one minute intervals using a CAN transmitter with temperature and relative humidity probes (G.Lufft Mess- und Regeltechnik GmbH, 70719 Fellbach, Germany).

The concentrations of oxygen (Oxygen analyzer type PMA-10, M&C Products Analysentechnik GmbH, Ratingen-Lintorf, Germany) and carbon dioxide (GM70 probe with MI70 read-out unit, Vaisala, Helsinki, Finland) in the test atmosphere were measured two times for each group, once during the first and once during the last week of the exposure period.


Prior to the first exposure, the output of the flame ionization detector of each TCA was dynamically calibrated. To this end, test atmospheres were generated at known concentrations by evaporating a flow of liquid test material, controlled using by a motor-driven syringe pump (WPI Type SPLG110, World Precision Instruments, Sarasota FL, USA) in a mass flow controlled (Bronkhorst Hi Tec) stream of compressed dry air, by directing it through a stainless steel tube which was kept at an above-ambient temperature to facilitate the evaporation (25-37°C) using a water mantle. The resulting test atmosphere was led directly to the TCA; excess air was exhausted. The mass flow controller was calibrated using a volumetric flow meter (DryCal, Bios International Corporation, Butler, NJ, USA) at the flow settings used for calibrating the TCA. The calibration settings were selected to generate about 80%, 100% and 120% of each target concentration. A zero calibration was included for each TCA, using clean dry air only. Linear relations were found between the response Y of the analyzers (in % of full scale) and the concentration X of the test material (in ppm; see below).

The calibrations were checked weekly during the study. To this end, a test atmosphere was generated at each target concentration as described above. The concentration as calculated from the measured flow of the mass flow controller and the settings of the pump, was compared to the concentration as measured by the analyzers. 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.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The overall average actual concentrations (± standard deviation) of nitromethane in the test atmospheres, as determined by total carbon analysis, were 303 (± 3.3), 601 (± 12.4) and 1178 (± 43.9) ppm for the low-, mid-, and high concentration, respectively (Table 1.1). These concentrations were close to the respective target concentrations of 300, 600 and 1200 ppm.
Details on mating procedure:
Upon arrival, the animals were housed in quarantine and checked for overt signs of ill health and anomalies. During the quarantine period, serological investigation of the microbiological status were carried out in a few randomly chosen rats of the lot delivered. Upon satisfactory serology results, the animals were transferred to their definitive animal room. The rats were acclimatized to the laboratory conditions for at least 5 days prior to the start of the exposure. At the end of the acclimatisation period, males and females were mated (two females were caged with one male). Animals were caged together until mating occurred. 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 (GD 0). Upon evidence of copulation the females were caged individually. Ninety-six mated females out of 104 females were distributed in an unbiased manner by manual randomization over the 4 experimental groups (24 mated females/group) in such a way that the animals from the same day of pregnancy were equally distributed over all groups. Females mated by the same male were placed in different groups. The remaining females were discarded after mating.
Duration of treatment / exposure:
The study comprised 4 groups of 24 mated females each, viz. one control group and three test groups receiving different concentrations of test material from gestation day (GD) 6 up to and including GD 20 (weekend and holidays included). The animals of the control group were handled in the same manner as those of the other groups, except for exposure to the test substance.
Frequency of treatment:
Animals were exposed for 6 hours per day from GD 6 up to and including GD 20.
Duration of test:
Exposure from gestation day (GD) 6 up to and including GD 20.
Doses / concentrationsopen allclose all
Dose / conc.:
0 ppm (nominal)
Dose / conc.:
300 ppm (nominal)
Dose / conc.:
600 ppm (nominal)
Dose / conc.:
1 200 ppm (nominal)
No. of animals per sex per dose:
In each group 24 females were used.
Control animals:
yes, sham-exposed
Details on study design:
DOSE SELECTION RATIONALE
In a dose range finding study for the OECD 414 study, mated females were exposed to concentrations of 25, 100 and 300 ppm nitromethane from gestation day 6 to gestation day 20. No treatment-related effects were observed in this study. In a subsequent main prenatal development study, mated females were exposed to concentrations of 0, 100, 300 and 600 ppm nitromethane from gestation day 6 to gestation day 20. No treatment-related effects were observed in this study on maternal parameters (body weight gain, food consumption, clinical observations) and litter parameters (number and distribution of live and dead fetuses, resorptions).

In a sub-chronic inhalation study with nitromethane, Fischer 344 rats were exposed to concentrations of 94, 188, 375, 750 and 1500 ppm, 6 hours per day, 5 days per week for 13 weeks. All rats survived to the end of the study. The final mean body weight and weight gain of male rats in the 1,500 ppm group were significantly less than those of the controls. Clinical findings included hindlimb paralysis in rats in the 750 and 1,500 ppm groups. In rats, inhalation exposure to nitromethane resulted in an exposure concentration-dependent, microcytic, responsive anemia; anemia was most pronounced in males and females exposed to 375 ppm or greater. On day 23, transient decreases in serum triiodothyronine, thyroxine, and free thyroxine were observed in male rats exposed to 375 ppm or greater and female rats exposed to 750 or 1,500 ppm. There was little or no pituitary response to the thyroid hormone decreases, as evidenced by the lack of significantly increased concentrations of thyroid-stimulating hormone in exposed rats. No biologically significant differences in organ weights were observed. The forelimb and hindlimb grip strengths of males in the 1,500 ppm group were significantly less than those of the controls. The hindlimb grip strengths of females in the 750 and 1,500 ppm groups were also significantly less than the control value. The NOAEL for systemic effects was 94 ppm in rats.

Following Haber’s rule, based on an No-Effect-Level of 94 ppm in the 90-day study, the No-Effect-Level in the 14-day exposure period in the OECD 414 study in rats would be expected around 600 ppm. Exposure to 600 ppm did not result in maternal effects in terms of body weight, food consumption or clinical signs. Taking into account both these results and Haber’s rule, a concentration of 600 ppm was selected as mid concentration level. As high concentration a level of 1200 ppm was selected, whereas 300 ppm was selected as low concentration level.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS
Each animal was observed daily in the morning hours (pre-dosing) by cage-side observations and, if necessary, handled to detect signs of toxicity. The animals were also observed about halfway through the 6-hour exposure period, in particular to monitor any breathing abnormalities and restlessness. All animals were also thoroughly checked again after exposure. All abnormalities, signs of ill health, reaction to treatment and mortality were recorded.

BODY WEIGHT
Body weights of the parental female animals was recorded on gestation days (GD) 0, 6, 9, 12, 15, 18 and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE
The food consumed for each mated female was measured over the periods: GD 0-6, GD 6-9, GD 9-12, GD 12-15, GD 15-18 and GD 18-21. The results are expressed in gram per animal per day.

POST-MORTEM EXAMINATIONS:
The females were killed by exsanguination after isofluorane anaesthesia on GD 21 and examined for gross abnormalities. The female showing signs of premature delivery was also examined macroscopically, and the number of fetuses, corpora lutea and implantations were recorded.
Blood was collected from all dams at necropsy and plasma (using heparin as anticoagulant) and serum samples were stored in a freezer (≤-18°C) for possible future analysis. The samples will be discarded after authorization of the report.

From all animals the following organs were preserved in formalin for possible future histopathological examination:
- nose
- larynx
- trachea
- lungs
- liver
- sciatic nerve
- gross lesions

All females killed on GD 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
- gross evaluation of placentas.
- abnormal tissues or organs in dams

The following organs were weighed:
- kidneys
- liver
- uterus, containing placentas and fetuses
- uterus, empty
- ovaries
- live fetuses (individually) with corresponding placentas

If necessary the implantation sites were made visible following Salewski E. (1964).
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: yes
Examinations included:
- Gravid uterus weight: yes
- Number of corpora lutea: yes
- Number of implantations: yes
- Number of early resorptions: yes
- Number of late resorptions: yes
Fetal examinations:
Fetuses were examined for external alterations and sacrificed by hypothermia Subsequently, approximately half of the fetuses of each litter was fixed in Bouin's fixative, examined for soft tissue anomalies according to a method modified after Barrow and Taylor (1969)5 and then discarded. Abnormal tissues were preserved in Bouin’s fixative. The other half of the fetuses were fixed in 70% alcohol, subsequently partly eviscerated, and then cleared in potassium hydroxide and stained with Alizarin Red S modified after Dawson (1926)6. They were examined for skeletal abnormalities and then retained. During the fetopathological examination, the observer was unaware of the dose group of the fetuses.

Indices:
The following parameters were calculated:
- 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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical abnormalities were noted during exposure. There were no treatment-related clinical abnormalities directly after exposure.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean body weight was statistically significantly lower in the high concentration group at the end of gestation (e.g. gestation days 18 and 21) as compared to the control group (13% reduction on day 21). Mean body weight changes were statistically significantly lower in the high concentration group in the intervals from gestation day 6-9, 15-18, 18-21 and also in the overall gestation period (38% reduction) as compared to the control group. No effects on body weight or body weight changes were observed in the low and mid concentration groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Mean food consumption was statistically significantly lower in the high concentration group as compared to the control group at the start of exposure (gestation days 6-8) and at the end of gestation (18-21). The reduction in food consumption was 11% on day 21. No effects on food consumption were observed in the low and mid concentration groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Mean terminal body weight was statistically significantly lower in the high concentration group as compared to the control group. The mean weight of the full uterus (including fetal and placental tissues) and empty uterus were statistically significantly lower in the high concentration group as compared to the control group. This was considered to be related to the increased number of late resorptions observed in this group. No effects on uterus weight were observed in the low and mid concentration groups.

Mean absolute ovary weight in the high concentration group was comparable to the control group, whereas relative ovary weight was statistically significantly increased. Mean absolute liver weight in the high concentration group was comparable to the control group, whereas relative liver weight was statistically significantly increased. The statistically significant differences in relative ovary weight and liver weight in the high concentration group are related to the lower terminal body weight in that group and are not considered to be treatment-related effects.

Mean absolute kidney weight was statistically significantly increased in all treatment groups and the effect was considered concentration-dependent. Therefore, the kidney was considered to be a target organ. Considering the limited increase in mean absolute kidney weight and the absence of statistically significant changes in relative kidney weight, the effect was not considered adverse in the low and mid concentration groups . In the high concentration group relative kidney weight was statistically significantly increased. This was considered related to the lower mean terminal body weight in this group. Therefore, the effect was considered not adverse.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
No effects were observed on the mean number of corpora lutea and the mean number of implantation sites.
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
No effects were observed on the mean pre-implantation loss. The mean post-implantation loss was statistically significantly increased in the high concentration group.
Total litter losses by resorption:
effects observed, treatment-related
Description (incidence and severity):
Total litter loss was observed in 5 out of 22 pregnant females of the high concentration group.
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
The mean number of early resorptions and dead fetuses was comparable in all groups. The mean number of late resorptions was statistically significantly increased in the high concentration group.
Dead fetuses:
no effects observed
Description (incidence and severity):
The mean number of dead fetuses was comparable in all groups.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
not examined
Other effects:
not examined

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
600 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: no effects observed at this level
Key result
Dose descriptor:
LOAEC
Effect level:
1 200 ppm (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake

Maternal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
uterus
Description (incidence and severity):
The mean weight of the full uterus (including fetal and placental tissues) and empty uterus were statistically significantly lower in the high concentration group as compared to the control group. This was considered to be related to the increased number of late resorptions observed in this group. No effects on uterus weight were observed in the low and mid concentration groups.

Results (fetuses)

Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean fetus weight was statistically significantly decreased in fetuses in the high concentration group, for both male and females fetuses. This effect is considered to be related to treatment. No effects on fetus weight were observed in the low and mid concentration groups.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not examined
Reduction in number of live offspring:
effects observed, treatment-related
Description (incidence and severity):
The reduction in the number of live offspring was statistically significantly decreased in fetuses in the high concentration group.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
effects observed, treatment-related
Changes in postnatal survival:
not examined
External malformations:
effects observed, treatment-related
Description (incidence and severity):
One fetus in the high concentration group showed sub-cutaneous oedema, which is classified as a malformation. One fetus in the control group and one in the high concentration group showed subcutaneous hemorrhageshaemorrhages. In addition, 13 fetuses in the high concentration group were pale. This was a statistically significant increased number as compared to the control group. No external malformations and variations were observed in the low and mid concentration groups.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
Observations in skeletal examinations indicated a delay in ossification, which was considered to be related to the smaller fetus size. In addition, the number of fetuses showing wavy ribs or bent radius and ulna bones were increased in the high concentration group. Wavy ribs and bending of the long bones are commonly observed skeletal variations in this strain of rats and are considered to be skeletal observations that are secondary to developmental toxicity.
Visceral malformations:
no effects observed
Other effects:
no effects observed

Effect levels (fetuses)

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
600 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects observed at this level
Key result
Dose descriptor:
LOAEC
Effect level:
1 200 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reduction in number of live offspring
fetal/pup body weight changes
changes in litter size and weights
changes in postnatal survival
external malformations

Fetal abnormalities

Key result
Abnormalities:
effects observed, treatment-related
Localisation:
skeletal: sternum
skeletal: hindlimb

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
1 200 ppm (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the lower body weight and body weight gain at the end of gestation and the lower feed intake at the start of exposure and the end of gestation in the high concentration group, the No Observed Adverse Effect Concentration (NOAEC) for maternal toxicity was considered to be 600 ppm. Based on the effects on post-implantation loss (as evidenced by an increase in late resorptions), fetus weight, the delay in ossification and skeletal and visceral observations in the high concentration group, the NOAEC for developmental toxicity was considered to be 600 ppm.
Executive summary:

The objective of this study was to provide data on the possible effects of the test material nitromethane on pregnant female rats (Wistar Han IGS), and on the development of the embryo and fetus. The test material was administered by inhalation (via whole body exposure) to groups of 24 mated females from gestation day (GD) 6 up to and including GD 20. The overall average actual concentrations of the test material in the test atmospheres as determined by total carbon analysis were 303 (± 3.3), 601 (± 12.4) and 1178 (± 43.9) ppm for the low-, mid- and high concentration, respectively and close to the respective target concentrations of 0, 300, 600 or 1200 ppm. At gestation day 21 caesarean section was performed, dams were examined macroscopically and reproductive organs, liver and kidney were weighed. Fetuses and placentas were weighed and fetuses were examined externally. Half of the fetuses of each litter were subjected to visceral examination, the other half to skeletal examination.

 

Daily whole body exposure (6 hours per day) to nitromethane from gestation day 6 up to and including gestation day 20 at concentrations of 0, 300, 600 and 1200 ppm resulted in:

-     No treatment related clinical signs or macroscopic changes in the dams;

-     Lower food consumption at the onset of treatment and at the end of gestation in the 1200 ppm group;

-     Lower body weight and body weight gain at the end of gestation in the 1200 ppm group;

-     Lower mean uterus weight in the 1200 ppm group, which was related to the increased post-implantation loss;

-     The kidney was identified as the target organ, as evidenced by a slight increase in kidney weight in all treatment groups. In view of the limited increase, this was not considered an adverse effect;

-     No effects on the mean number of corpora lutea or the mean number of implantation sites;

-     A marked, statistically significant, increase in post-implantation loss in the 1200 ppm group, due to a statistically significant increase in late resorptions and the complete litter loss of 5 females;

-     Statistically significantly decreased mean fetus weight in the 1200 ppm group.

-     Delayed ossification in the fetuses in the 1200 ppm group. In addition, the visceral observations and skeletal observations in the 1200 ppm group, were indicative for fetal toxicity or considered secondary to the fetal toxicity observed in this group.

-     No effects on fetal weight, visceral observations and skeletal observations were observed in the 300 and 600 ppm groups.

 

Based on the lower body weight and body weight gain at the end of gestation and the lower feed intake at the start of exposure and the end of gestation in the high concentration group, the No Observed Adverse Effect Concentration (NOAEC) for maternal toxicity was considered to be 600 ppm. Based on the effects on post-implantation loss (as evidenced by an increase in late resorptions), fetus weight, the delay in ossification and skeletal and visceral observations in the high concentration group, the NOAEC for developmental toxicity was considered to be 600 ppm.