Iodomethane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 April 2001 to 11 July 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

Draft

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: JMAFF 12 NouSan No. 8147

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Iodomethane

EC Number:

200-819-5

EC Name:

Iodomethane

Cas Number:

74-88-4

Molecular formula:

CH3I

IUPAC Name:

iodomethane

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): Iodomethane
- Physical state: liquid
- Analytical purity: 99.7%
- Impurities (identity and concentrations): 0.2 % water and <0.1 % methanol
- Composition of test material, percentage of components: not stated
- Isomers composition: not stated

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: six and one-half months old
- Weight at study initiation: 2989 to 4133 g
- Housing: individually housed in clean, stainless-steel wire-bottom cages suspended above ground corn cob bedding.
- Diet: ad libitum (restricted to 150 g/animal/day during the acclimation period)
- Water: ad libitum
- Acclimation period: 20 or 27 days

ENVIRONMENTAL CONDITIONS
- Temperature: 18.9 to 22.1 °C (66.0 to 71.7 °F)
- Humidity: 44.1 to 70.9 %
- Air changes: Air handling units were set to provide approximately 10 fresh air changes per hour.
- Photoperiod: Light timers were calibrated to provide a 12-hour light/12-hour dark photoperiod.

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

EXPOSURE METHODS
- Because of the limited amount of space available in the exposure chambers, animals in each group were divided into two replicates. The exposures of each replicate of animals were conducted separately, with the initial exposure of animals in the second replicate occurring four weeks following the initial exposure of animals in the first replicate.
- All animals in each replicate were exposed simultaneously in four 1.5 m^3 stainless steel and glass whole-body exposure chambers. One chamber was dedicated for each group for the duration of exposures.
-The test material and filtered air were administered as daily six-hour exposures during a period that included major organogenesis, gestation days 6 through 28 (from implantation to one day prior to laparohysterectomy). All rabbits were exposed at approximately the same time each day, and exposures were conducted seven days per week. Animal cage batteries were rotated through the different cage rack positions within the chamber on a daily basis to minimize the effects of any potential variation due to chamber position. The control group was exposed to filtered air under conditions identical to those used for the test material exposure groups.
- The rabbits were removed from their home cages, placed in exposure caging in the animal room and transported to the exposure chambers for exposure to the test material. The animals were exposed for the requisite duration and then returned to their home cages. Food and water were withheld during each daily exposure period.
-The daily mean chamber temperatures were 72-77 °F (22-25 °C). The daily mean chamber humidity ranged from 42-76 %.
- Exposure concentrations within each chamber were measured at least 10 times (approximately every 35 minutes) during each daily exposure period by a validated gas chromatographic method.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

NOMINAL EXPOSURE CONCENTRATIONS
- A nominal exposure concentration was calculated for each daily exposure for each chamber from the total amount of test material used during the exposure and the total volume of air passed through the chamber during that day's exposure. The amount of test material used was obtained by weighing the resevoir containing the test material for each chamber prior to and after each daily exposure. The total volume of air passed through each chamber was calculated from the daily average chamber ventilation flow rate in litres per minute (LPM) and the exposure duration. The nominal concentration was calculated as follows:
ppm Iodomethane = (Wt. Iodomethane · Mol Vol · 10^6) / (MW. Ch.Flow . Exp.Dur)
Where:
Wt. Iodomethane = weight of test material in grams
Mol Vol = Molar volume at 730 mmHg and 21 °C, 25.11 L/mole
10^6 = ppm conversion factor
MW = molecular weight, 141.94 g/mole
Ch. Flow = Daily average chamber flowrate for a given day, in LPM.
Exp. Dur. = Duration of a given day's exposure, in minutes

ACTUAL EXPOSURE CONCENTRATIONS
- Actual exposure concentrations were measured using a gas chromatograph (GC). Samples of the exposure atmospheres from each chamber were automatically collected at approximately 35-minute intervals using a sample loop and computer-controlled gas-sampling and multiposition valve. The following summarises the GC conditions:
Instrument: Hewlett Packard 5890 Series II with a 3396 Series II integrator
Detector: Flame ionisation
Column: J & W DB-Wax Narrow Bore, 30 m x 0.25 mm I.D., 0.25- micron film thickness
Gases: (Pressure (psig) Flow Rate (mL/min.)): Carrier - Helium 46 15.4, Fuel - Hydrogen 18 31, Air 35 300
Temperatures (°C): Injector 250, Column 45, isothermal and Detector 250
Injection volume (mL) 0.25
Retention time (min.) Approximately 1.0 min.
Integrator Run Parameters: Chart Zero Offset 0, Chart Attenuation 1, Chart Speed 2.5 cm/min, Peak Area Rejection Value 0, Peak Threshold 2 and Peak Width 0.04
- The chromatograph was standardised using 40-liter Tedlar® gas bags prepared to contain known concentrations of the test material. The standard bags were prepared by injecting known volumes of test material into a 500 mL glass vaporisation bulb. A continuous flow of air carried the vaporised test material to a 40 L bag. The total volume of air was measured by a dry test meter (Model DTM-200A, American Meter Co., Nebraska City, PA). Concentrations of the gas-phase standards were calculated as follows: Concentration = (VOL · R · T · D · 10^-3 · 10^6) / (L · GMW · P)
Where:
Conc. is in ppm
VOL = volume of test material vaporised into bag in μL
R = universal gas constant, 62.36 L mmHg/mole K
T = nominal laboratory temperature in K (273 + 21 °C = 294 K)
D = density of the test material, 2.280 g/mL
L = volume of air used to prepare bag, 32 L
GMW = gram molecular weight, 141.94 g/mole
P = nominal laboratory barometric pressure, 730 mmHg
10^-3 = μL to mL conversion factor
10^6 = conversion factor to ppm

-Standards: 32 L of air was added to the following to prepare the standards: 1.3 ppm: 0.1 µL test material, 6 ppm: 0.5 µL test material, 13 ppm: 1.0 µL test material, 21 ppm: 1.7 µL test material and 29 ppm: 2.3 µL test material
Each standard was prepared in triplicate prior to the exposure period and analysed with the GC. A least-squares line was fitted to the resulting peak areas. Concentrations were then calculated using the slope and intercept of this prime calibration curve. On a daily basis, the integrity of the prime calibration curve was checked by analysing one freshly prepared standard. On a rotational basis, a different one of the five standards was used each day. If the analysed concentration was within ± 20 % for the 1.3 PPM standard, ± 15 % for the 6 PPM standard, and ± 10 % for the remaining standards of the known concentration, the GC was considered within calibration specifications.

DETERMINATION OF HOMOGENEITY OF EXPOSURE ATMOSPHERES
- Evaluation of the homogeneity of exposure concentrations was accomplished during the method development phase of the study prior to animal exposures. Four test locations and a reference location were used for these determinations. The test locations were Right Upper Front, Right Lower Rear, Left Lower Front, Left Upper Rear identified as 1, 2, 3 and 4, respectively for Chamber 4. The test locations for Chambers 2 and 3 were Right Upper Rear, Left Lower Rear, Right Lower Front and Left Upper Front, identified as 1, 2, 3 and 4, respectively. Samples were collected as rapidly as possible always collecting a sample from the reference location and then from one of the four test locations. For each test location, the measured concentration was calculated as a percent difference from the reference location. The homogeneity determination was performed in triplicate for each test material exposure chamber
- Results indicated that homogeneity of exposure atmospheres were adequate for the purpose of this study. Maximum mean % from reference was – 9.5 %.

RESULTS
- Nominal Exposure Concentrations: 4.7, 16 and 28 ppm
- Actual Exposure Concentrations: 2, 10 and 20 ppm

Details on mating procedure:

- Semen was collected from eight resident male rabbits of the same strain and obtained from the same source as the females. The collection apparatus consisted of an artificial vagina filled with warm water (approximately 55°C) and fitted with a graduated collection tube. After collection, the gelatinous plug (if any) was removed from each sample and the ejaculate was evaluated for volume, motility and concentration.
- Motility was determined microscopically on a percentage basis from a drop slide preparation using low-power magnification. The concentration of sperm/mL was determined using a standard dilution in a red blood cell pipette and a haemocytometer under high-power magnification. Semen with greater than 50 % motility was diluted with 3.0 mL of 0.9 % USP physiological normal saline and was maintained at 34-37 °C in a water bath during the insemination procedure. The final concentration obtained was greater than 3 million motile sperm/mL.
- Diluted semen from one male was used to inseminate two or four females in each group (a maximum of two females in each replicate in each group was inseminated with semen from one male). A 0.25- to 0.50-mL aliquot of each diluted semen sample was deposited into the anterior vagina of each female with a glass insemination pipette. Immediately following insemination, each doe was administered an intravenous injection (100 USP Units) of human chorionic gonadotropin via the marginal ear vein to induce ovulation.
- The day of insemination was designated gestation day 0.

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

once daily

Duration of test:

Gestation days 6 to 28

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 ppm (analytical)

Doses / concentrations 2

Dose / conc.:

10 ppm (analytical)

Doses / concentrations 3

Dose / conc.:

20 ppm (analytical)

No. of animals per sex per dose:

24 females per dose

Control animals:

yes

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- All rabbits were observed twice daily, once in the morning and once in the afternoon (at least seven hours apart), for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Individual detailed clinical observations were recorded from day 0 through 29 of gestation (prior to test article administration during the exposure period).
- Animals were observed for signs of toxicity at the midpoint of exposure and within one hour following exposure; all significant clinical findings were recorded at these observation periods.

BODY WEIGHT: Yes
- Individual maternal body weights were recorded on gestation days 0 and 6-29 (daily). A group mean body weight was calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-15, 15-21, 21-29, 6-29 and 0-29.
- Gravid uterine weight was collected and net body weight (the day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the day 0-29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

FOOD CONSUMPTION: Yes
- Individual food consumption was recorded on a daily basis throughout gestation. Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.

GESTATION DAY 29 LAPAROHYSTERECTOMY
- All surviving females were euthanised on gestation day 29 by an intravenous injection of sodium pentobarbital via the marginal ear vein. The thoracic, abdominal and pelvic cavities were opened by a ventral midline incision and the contents examined. In all instances, the post mortem findings were correlated with the ante mortem comments and any abnormalities were recorded. The uterus and ovaries were excised and the number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all foetuses, early and late resorptions and the total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
- Maternal tissues were preserved in 10 % neutral-buffered formalin for possible future histopathological examination only as indicated by the gross findings. The carcass of each female was then discarded. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulphide solution for detection of early implantation loss.

Ovaries and uterine content:

GESTATION DAY 29 LAPAROHYSTERECTOMY
- The uterus and ovaries were excised and the number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all foetuses, early and late resorptions and the total number of implantation sites were recorded. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
- Maternal tissues were preserved in 10 % neutral-buffered formalin for possible future histopathological examination only as indicated by the gross findings. The carcass of each female was then discarded. Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulphide solution for detection of early implantation loss.

Fetal examinations:

- Each foetus was weighed, tagged for identification, and euthanised by an intrathoracic injection of sodium pentobarbital, if necessary.
- A detailed external examination of each foetus was conducted to include, but was not limited to, an examination of the eyes, palate and external orifices, and each finding was recorded. Crown-rump measurements were recorded for nonviable foetuses and late resorptions, if present, the degree of autolysis was recorded, and the tissues were discarded. The sex of each foetus was determined internally and each foetus was examined viscerally by a fresh dissection technique to include the heart and major vessels. The brain from each foetus was examined by a mid-coronal slice. foetal kidneys were examined and graded for renal papillae development.
- All carcasses were eviscerated and fixed in 100 % ethyl alcohol. Following fixation in alcohol, each foetus was macerated in potassium hydroxide and stained with Alizarin Red. External, visceral and skeletal findings were recorded as developmental malformations (those structural anomalies that alter general body conformity, disrupt or interfere with body function, or may be incompatible with life) or variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity, representing slight deviations from normal).

Statistics:

All analyses were conducted using two-tailed tests for minimum significance levels of 5 and 1 %, comparing each treated group to the vehicle control group:
- One-way ANOVA with Dunnett’s test: Corpora Lutea, Total Implantations, Viable Foetuses, Foetal Body Weights, Maternal Body Weights, Body Weight Changes, Net Body Weights, Net Body Weight Changes and Gravid Uterine Weights and Maternal Food Consumption.
- Kruskal-Wallis test with Mann-Whitney U test: Litter Proportions of Intrauterine Data (Considering the Litter, Rather than the Foetus, as the Experimental Unit), Litter Proportions of Foetal Malformations and Developmental Variations.

Indices:

Group Mean Litter Basis:
- Postimplantation Loss/Litter = [No. Dead Foetuses, Resorptions (Early/Late)/Group] / No. Gravid Females/Group
- Proportional Litter Basis: Summation per Group (%) = [ΣPostimplantation Loss/Litter (%)*] / No. of Litters/Group
*= [[No. Dead Foetuses, Resorptions (Early/Late)/Litter ] / No. Implantation Sites/Litter] x 100

- The foetal developmental findings were summarised by: 1) presenting the incidence of a given finding both as the number of foetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected foetuses in a litter on a proportional basis as follows:
Summation per Group (%) = (Σ Viable Foetuses Affected/Litter (%)) / No. of Litters/Group
Viable Foetuses Affected/Litter (%) = [(No. Viable Foetuses Affected/Litter) / No. Viable Foetuses/Litter] x 100

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The only exposure-related clinical finding observed was wet clear matting around the nose, noted at increased frequencies during the daily examinations (conducted prior to daily exposures) in all exposure groups relative to the control group. The matting was generally slight and was noted predominantly during the last week of exposure. This finding was considered to be exposure-related, but was not considered to be a sign of toxicity. It is acknowledged that wet clear matting around the nose was also observed in the control group, but the finding was noted less frequently and in fewer animals than in the exposed groups. Other clinical findings noted in the exposed groups, including hair loss, scabbing and staining on various body surfaces, soft stool and decreased defecation, occurred infrequently and/or at similar frequencies in the control group.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

All females survived to the scheduled laparohysterectomy on gestation day 29.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Mean maternal body weight changes in the 20 ppm group were similar to the control group values for gestation days 6-9, 9-15 and 15-21. During gestation days 24-29, a statistically significant (p<0.05) mean body weight loss was observed in this group. When the entire exposure period (gestation days 6-29) was evaluated, mean body weight gain in the 20 ppm group was reduced relative to the control group value; the difference was statistically significant (p<0.05). Mean body weights throughout gestation, net body weight and net body weight gain were unaffected by exposure to the test material in the 20 ppm group.
- In the 10 ppm group, mean body weights and body weight gains throughout gestation were unaffected by exposure to the test material. Differences from the control group values were slight and were not statistically significant. Mean gravid uterine weight in the 10 ppm group was slightly reduced (not statistically significant) relative to the control group value. This reduction was attributed to a decrease in the mean number of viable foetuses in this group. Mean net body weight and net body weight gain in the 10 ppm group were unaffected by test material exposure.
- Mean body weights and body weight gains throughout gestation, gravid uterine weight, net body weight and net body weight gain in the 2 ppm group were unaffected by exposure to the test material. Differences from the control group were slight and were not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Group mean food consumption, evaluated as g/animal/day and g/kg/day, throughout gestation was unaffected by test material exposure at exposure levels of 2, 10 and 20 ppm. Food consumption was slightly reduced from gestation days 24-29 in a few of the 10 and 20 ppm maternal animals which had one or more late resorptions at the post mortem examination. These reductions were not of significant magnitude to effect the group mean value. Sporadic statistically significant (p<0.05 or p<0.01) increases in food consumption were noted in the 2 ppm group; however, because similar increases were not observed at exposure levels of 10 and 20 ppm, the increases were not attributed to exposure.

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:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

-No exposure-related internal findings were noted at the scheduled necropsy on gestation day 29. Cystic oviducts were noted in 3, 1, 7 and 4 animals in the control, 2, 10 and 20 ppm groups, respectively. Accessory spleens were observed in 4, 2, 4 and 4 females in the same respective dose groups. A major blood vessel variation (the left carotid artery arose from the brachiocephalic trunk) was observed in one female in the 20 ppm group. One female in the 2 ppm group had a mass in the median lobe of the liver. No other internal findings were observed at the scheduled necropsy.

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:

not examined

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

- The mean litter proportion of post-implantation losses was increased in the 20 ppm group relative to the control group values; the increases were statistically significant (p<0.05 or p<0.01). The mean litter proportion of post-implantation losses was also increased relative to the maximum mean values in the WIL historical control data. These increases in post-implantation losses resulted in statistically significant (p<0.01) reductions in the mean litter proportion and mean number of viable foetuses in the 20 ppm group.
- In the 10 ppm group, increases (not statistically significant) were observed in the mean litter proportion of post-implantation losses. The value was higher than the mean values in the WIL historical control data. The increase in post-implantation loss resulted in a reduction (not statistically significant) in the mean number of foetuses in this group.
- No effects of the test material were observed on intrauterine parameters in the 2 ppm group.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

The mean litter proportion of total resorptions was increased in the 20 ppm group relative to the control group values; the increases were statistically significant (p<0.05 or p<0.01).

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

- The mean litter proportion of late resorptions was increased in the 20 ppm group relative to the control group values; the increases were statistically significant (p<0.05 or p<0.01). The mean litter proportions of late resorptions was also increased relative to the maximum mean values in the WIL historical control data.
- In the 10 ppm group, and increase (not statistically significant) was observed in mean litter proportion of late resorptions. The value were higher than the mean value in the laboratory's historical control data (the mean litter proportion of late resorptions was also higher than the maximum mean value in the WIL historical control data). It should be noted that late resorptions in the 10 ppm group were noted more frequently in the first replicate than in the second replicate of animals.
- No effects of the test material were observed on intrauterine parameters in the 2 ppm group.

Dead fetuses:

not examined

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)

Key result

Dose descriptor:

NOAEL

Effect level:

10 ppm (analytical)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Mean foetal body weights (male, female and combined) were reduced in the 20 ppm group relative to the concurrent control group values; the differences were statistically significant (p<0.01). The combined value was also reduced relative to the minimum mean value in the WIL historical control data.
- Mean foetal body weights (male, female and combined) were reduced in the 10 ppm group relative to the control group values; the difference for the female value was statistically significant (p<0.05). Mean numbers of foetuses and mean combined foetal body weights were also reduced relative to the mean values in the WIL historical control data (although the mean foetal body weight value was within the range of the historical control data). When evaluating the individual data, mean foetal weight values for surviving foetuses from litters which expressed a high degree of late embryonic resorption were often lower than the group mean value.
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):

Mean numbers of foetuses awere reduced relative to the mean values in the WIL historical control data.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No effect of test material exposure at an exposure level of 20 ppm was observed on the foetal sex ratio.

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- External malformations were observed in 1(1), 2(2) and 1(1) foetuses (litters) in the control, 10 and 20 ppm groups, respectively. One foetus in the 20 ppm group had tarsal flexure. One foetus in the 10 ppm group had a disseminated subcutaneous haemorrhage in the dorsal posterior area. One foetus in the 10 ppm group had a bent medial tail (only 11 caudal vertebrae were present, and caudal vertebrae nos. 7 through 10 were fused and/or misshapen). One foetus in the control group had a small nose (the rostral portion of the nasal bones was fused).
- No external developmental variations were observed for foetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- Skeletal malformations were observed in three foetuses in the 2 ppm group and in one foetus in the 10 ppm group. One foetus in each of these groups had vertebral anomalies with associated rib anomalies; these anomalies consisted of malpositioned arches and centra, fused centra and ribs, extra ribs, an extra arch, a small arch and an absent centrum. One foetus in the 2 ppm group had severe malalignment of sternebrae nos. 2 through 5. One foetus in this litter had fusion of sternebrae nos. 4 and 5.
- Skeletal developmental variations observed in all exposure groups included 13th full ribs, 13th rudimentary ribs, 27 presacral vertebrae and bent hyoid arches. Other skeletal developmental variations noted in the exposed groups occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- No soft tissue malformations were observed for foetuses in this study.
- Soft tissue developmental variations that were observed at all exposure levels included major blood vessel variations (either the left carotid artery arose from the brachiocephalic trunk or the right carotid and subclavian arteries arose independently from the aortic arch [no brachiocephalic trunk was present]). Accessory spleens were also observed at all exposure levels; the mean litter proportion of accessory spleens in the 10 ppm group was reduced (statistically significant at p<0.05) relative to the control group value. The reduction did not occur in a manner that was dose-related, and it was not attributed to test material exposure. Four and five foetuses in the 2 and 10 ppm groups, respectively, had small gallbladders; the mean litter proportion of small gallbladders was significantly (p<0.05) increased in the 10 ppm group relative to the control group value. Because the increase did not occur in a manner that was dose-related, it was not attributed to test material exposure. One foetus in the 10 ppm group had an undeveloped right renal papilla. Three foetuses in the 2 ppm group had retrocaval ureters. One foetus each in the control and 2 ppm groups had small spleens. A haemorrhagic ring was observed around the right iris of a single control group foetus. Because the findings in the exposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related, they were not attributed to test material exposure.
- Several soft tissue findings were observed that were not classified as either malformations or developmental variations and were not included in any tabulation. Thyroid glands that were dark red and/or enlarged were observed in four foetuses in the 10 ppm group. Reddened thymus glands were observed in one foetus each in the 2 and 10 ppm groups. A cystic ovary was observed in one foetus in the 20 ppm group. One foetus in the 2 ppm group had a white area on the liver, and one foetus in the control group had a cystic liver. Because these findings occurred in a manner that was not dose-related or occurred primarily in single foetuses or litters, they were not attributed to test material exposure.

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

2 ppm (analytical)

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

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Selected caesarean section data

(ppm)	0	2	10	20
No. in gp	24	24	24	24
Late resorptions (%/litter)	1.7(23)	3.1(20)	11.1(20)#	21.5(21)**#
Late resorptions (%/litter) laboratory’s historical control range = 0.0 – 6.2
Post implantation loss (%/litter)	13.8(23)	12.1(20)	17.9(20)	32.3(21)**#
Post implantation loss (%/litter) laboratory’s historical control range = 0.6 – 23.1
Proportion of viable foetuses (%/litter)	86.2(23)	87.9(20)	82.1(20)	67.7(21)**#
Viable foetuses (%/litter) laboratory’s historical control range = 76.9 – 99.4
Mean number of viable foetuses/dam	6.1	5.5	4.6	3.6**#
Viable foetuses/dam laboratory’s historical control range = 4.74 – 8.3
Mean foetal weight (g)	47.0(22)	45.8(19)	43.3(20)	37.8(20)**#
Mean foetal weight (g) laboratory’s historical control range = 39.2 – 51.8

* Statistically significant p<0.05); **Statistically significant (p<0.01); # Outside of laboratory’s historical control range

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study it was concluded that the NOAEL for maternal toxicity was 10 ppm and the NOAEL for prenatal developmental toxicity was 2 ppm.

Executive summary:

The potential maternal toxicity and prenatal developmental toxicity of the test material were evaluated in accordance with the standardised guidelines OECD 414, OPPTS 870.3700 and JMAFF 12 Nousan No. 8147, under GLP conditions.

The test material was administered via whole-body inhalation as a vapour to three groups of 24 artificially inseminated New Zealand White rabbits once daily for a period of six hours per day from gestation days 6 through 28. A concurrent control group, composed of 24 artificially inseminated rabbits, was exposed to clean, filtered air on a comparable regimen. Because of the limited amount of space available in the exposure chambers, each group was divided into two replicates. Artificial insemination of animals in each replicate was conducted over two days, with four weeks between replicates. The targeted exposure concentrations were 2, 10 and 20 ppm. The overall test atmosphere concentrations were measured using gas chromatography and were found to be 2, 10 and 20 ppm for each replicate. Clinical observations, body weights and food consumption were recorded. On gestation day 29, a laparohysterectomy was performed on all rabbits. The uteri and ovaries were examined and the numbers of foetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and the net body weight changes were calculated. Foetuses were weighed, sexed and examined for external, soft tissue and skeletal malformations and developmental variations.

All females survived to the scheduled laparohysterectomy on gestation day 29. Wet clear matting around the nose was observed at increased frequencies during the daily examinations in all exposure groups relative to the control group; while this finding was considered to be exposure-related, it was not considered to be a sign of toxicity. No exposure-related internal findings were noted at the scheduled necropsy.

In the 20 ppm group, mean body weight changes were similar to the control group values for gestation days 6-9, 9-15 and 15-21. During gestation days 24-29, a statistically significant mean body weight loss was observed in this group. Mean body weights throughout gestation, net body weight and net body weight gain in the 20 ppm group were unaffected by test material exposure. In the 10 ppm group, a slight (not statistically significant) reduction was observed in the mean gravid uterine weight. In the 2 and 10 ppm groups, mean body weights and body weight gains throughout gestation, net body weight and net body weight gain were unaffected by test material exposure. No effect of test material exposure was observed on mean gravid uterine weight in the 2 ppm group. Group mean food consumption, evaluated as g/animal/day and g/kg/day, was unaffected by test material exposure at all exposure levels throughout gestation. Food consumption was slightly reduced from gestation days 24-29 in a few of the 10 and 20 ppm maternal animals which had one or more late resorptions at the post mortem examination. These reductions were not of significant magnitude to effect the group mean value.

In the 10 and 20 ppm groups, increases were noted in mean litter proportions of late resorptions and postimplantation losses; the increases in the 20 ppm group were statistically significant relative to the control group values. The increases in mean litter proportions of late resorptions in both of these groups were higher than the maximum mean values in the WIL historical control data. These increases resulted in reduced (statistically significant for the 20 ppm group) mean numbers of foetuses in the 10 and 20 ppm groups. Mean foetal body weights (male, female and combined) were reduced in the 10 and 20 ppm groups; the reductions were statistically significant in both sexes in the 20 ppm group but only in female foetuses at 10 ppm. The mean foetal weight in the 10 and 20 ppm groups were 8 and 20 % lower, respectively, than the concurrent control value. The decrement in foetal body mass at term in the high exposure group was below the range of the WIL historical control data sets. In contrast, the value for the 10 ppm group was within the range of the historical control data. Thus, the effect at 10 ppm was interpreted as treatment-related, but the toxicological significance is considered equivocal. No effects of test material exposure were observed on intrauterine parameters in the 2 ppm group.

Foetuses (litters) available for morphological evaluation numbered 140(22), 109(19), 91(20) and 76(20) in the control, 2, 10 and 20 ppm groups, respectively. Malformations were observed in 1(1), 3(2), 3(3) and 1(1) foetuses (litters) from the same respective dose groups, and were considered to be spontaneous in origin. foetal developmental variations in the exposed groups occurred infrequently or at similar frequencies in the control group; no relationship to test material exposure was evident.

In conclusion, maternal toxicity was expressed in the 20 ppm group by reduced mean body weight gains or mean body weight losses. Prenatal developmental toxicity was expressed at exposure levels of 10 and 20 ppm by increases in post implantation losses (primarily late resorptions), reduced mean numbers of viable foetuses and/or reduced mean foetal body weights. Under the conditions of this study, the NOAEL (no-observed-adverse-effect level) for maternal toxicity via whole-body inhalation exposure was considered to be 10 ppm, and the NOAEL for prenatal developmental toxicity was considered to be 2 ppm.