Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on developmental toxicity

Description of key information

Prenatal Developmental Toxicity Study - rats - inhalation

An OECD 414 study was performed to assess the influence of Methylal, a solvent, on pregnant animals and on embryo-fetal survival and development when administered during the

organogenesis and fetal growth phases of Specific Pathogen Free rats (Crt: CD® BR VAF/Plus strain).

Twenty-five sexually mature (8-10 weeks old) female rats which were time-mated to identified males of the same strain and supplied by Charles River UK Limited (Manston Road, Margate, Kent.Charles UK) were subjected by whole-body exposure, to the vapour of methylal. Exposure concentrations of 0 (Control), 386, 1954 and 10068 ppm were administered for six hours each day from Days 6 to 15 post coitum inclusive. On Day 20 post coitum, all rats were killed, subjected to post mortem examination, litter values were determined and the foetuses were examined.

Treatment-related effects were confined to the parent females exposed to 10068ppm and comprised lower bodyweight gain with a concomitant reduction in food intake and a markedly greater water intake. No treatment-related effects on the litters were observed.

Foetal pathological examination revealed no changes that were considered to be attributable to exposure to methylal.

The no effect exposure level for this study was 1954 ppm. However, it is concluded that exposure of the parent female from Days 6 to 15 of pregnancy to 10068 ppm methylal has no effect on embryofoetal development.

Prenatal Developmental Toxicity Study - rabbits - oral

An OECD 414 study was performed to assess the influence of Methylal, a solvent, on pregnant animals and on embryo-fetal survival and development when administered during the

organogenesis and fetal growth phases of the New Zealand White rabbit.

Three groups of 22 females received Methylal at doses of 100, 300 or 1000 mg/kg/day by oral administration, from Day 6 to 28 after mating. A similarly constituted Control group

received the vehicle, water for formulation, at the same volume dose as the treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal

examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus

weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Three animals died prior to scheduled termination:

Group 1 female no 11 was found dead on GD14; death attributed to accidental trauma during dose administration.

Group 2 female no 31 was found dead on GD19. Terminal signs included reduced food consumption but clinical condition appeared normal. There were no macroscopic findings

at necropsy; the cause of death remains unclear.

Group 4 female no 86 was killed prematurely with evidence of abortion on GD27. Terminal signs included reduced food consumption, slight weight loss and thin build with pink/red staining in the cage and on the cage tray paper. Macroscopic examination revealed fetal material in the stomach and a pale liver.

At routine physical examination there were a slightly higher number of females at 1000 mg/kg/day that showed reduced faecal output.

Females receiving 1000 mg/kg/day showed slightly but significantly greater weight loss during the first two days of treatment (GD6-8; p<0.01). From GD6 to GD16 females receiving 1000 mg/kg/day showed statistically significantly low food consumption when compared with the Controls; thereafter consumption was similar to Controls

Bodyweight gain and food consumption at 100 and 300 mg/kg/day were unaffected by treatment and the maternal weight change following adjustment for the gravid uterine weight showed no adverse effect of treatment at dose levels up to and including 1000 mg/kg/day.

There were no macroscopic findings on GD29 that could be attributed to treatment.

At 300 and 1000 mg/kg/day the mean number of resorptions and resultant post-implantation loss was slightly high when compared with Controls; with the difference attaining statistical significance at 1000 mg/kg/day (p<0.05). However, review of the individual data shows that this difference at 300 mg/kg/day can be attributed to a single female/litter with 75% post-implantation litter loss; when the data for this litter is excluded, the mean post-implantation loss is less than that of the low dose group. There was no adverse effect on the live litter size, mean fetal or placental weight.

Examination of fetuses at 100, 300 and 1000 mg/kg/day revealed low incidences of major cranial abnormalities (Acephaly, Anencephaly and Cranioschisis) with related eye/palate, cervical vertebral and limb abnormalities. However, the observed cranial malformations are located in separate anatomic locations, undergo disparate morphogenetic processes, and occur at different times in gestation. Therefore, they are not considered related findings.

At 1000 mg/kg/day, fetal skeletal examination revealed an increased incidence of full supernumerary 13th rib, 20 thoracolumbar vertebrae, unilateral shift of pelvic girdle and a decrease in incidence of 7th costal cartilage not connected to sternum. These conditions are commonly observed skeletal variants in rabbits.

Therefore based on the body weight loss, low food consumption and increased post-implantation loss at 1000 mg/kg/day, the intermediate dose level of 300 mg/kg/day is considered to be both the maternal and fetal no observed adverse effect level (NOAEL).

QSAR predictions

QSAR prediction of developmental toxicity to dimethoxymethane using the Leadscope structural dysmorphogenesis in rat, rodent, mouse and rabbit show negative predictions. Different routes of exposure were used in the creation of the training sets used in these models.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 1996-January 1997
Reliability:
1 (reliable without restriction)
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Crl:CDRBRVAF/plus
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent, UK
- Age at study initiation: 8-10 weeks old
- Weight at study initiation: 175-291 g
- Fasting period before study: Not specified
- Housing: The animals were housed in group of four in suspended steel cages a cage equipped with solid sides and wire grid front, back and floor. Each cage measured 53 cm long, 35 cm long and 25 cm high. Plastic trays, lined with absorbent paper were placed below each cage to collect excreta. The tray paper was changed daily throughout the study. Each group of animals was kept in a separate ventilated area to prevent any possible cross contamination between groups once exposures had been commenced.
- Diet (e.g. ad libitum): All animals were given free access to special Diet Services (SDS) Laboratory Animal Diet No. 1 except during the 6 hour exposure period on Days 6 to 15 of presumed pregnancy inclusive.
- Water (e.g. ad libitum): All animals were given free access to tap water except during the 6 hour exposure period on Days 6 to 15 of presumed pregnancy inclusive.
- Acclimation period: Not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16-22°C
- Humidity (%): 28-60%
- Air changes (per hr): Not specified
- Photoperiod (hrs dark / hrs light): The lighting was controlled to give 12 hours light (07.30 to 19.30) and 12 hours dark per 24 hours.

IN-LIFE DATES: From: December23, 1996 To : January 6-8, 1996
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The exposure chambers were of stainless steel and glass construction and consisted of a cuboidal body fitted with a pyramidal base and top. Each chamber was approximately 0.75 m3 internal volume.
At the apex of the upper pyramidal figure was the tangentially mounted air duct. Immediately below this was a perforated canister that ensured equal distribution of the test atmosphere within the chamber.
Access to the chamber was through a hinged, glass panelled, stainless steel framed door at the front of the box section. The door was sealed using moulded rubber sealing strip.
Exposure cages constructed of stainless steel mesh were suspended on a framework arranged on four levels. Each level held four cages each capable of individually housing four rats. This gave a total animal exposure capacity of 64 rats. However, in this investigation a maximum of 4 cages were
used. A rat was placed in each of the front compartments of the exposure cage to facilitate ease of observation during exposure. The position of each individual within the chamber was recorded. Animal positions were changed daily throughout exposures to ensure that each rat spent time in each
position within the chamber.
A wet and dry mercury bulb thermohygrometer, used to monitor chamber temperature and relative humidity, was suspended in the chamber, visible through the glass panelled door.
The pyramidal base of each chamber was fitted with a 2 inch drain. The drain connected with a common drainage system via a ball valve.
A square tubular exhaust plenum, 3 inches in diameter and perforated along the ventral surface, was situated in the pyramidal base. This connected with the main extract system.

During exposure 5 it became apparent that insufficient test material remained for the remainder of the study. In order to conserve test material and allow exposures to continue to the planned termination, a 200 I Tedlar® gas bag was inflated and inserted into the High dose group chamber. The diluent air supply to the High dose group chamber was reduced to 85 Vmin such that the total airflow was 110 Vminthus maintaining 12 air changes per hour as required by the regulatory guidelines. This allowed the test substance feed rate to the High dose group chamber to be reduced and the exposures to continue nonnally. This modification of the chamber volume and airflow had no effect on the integrity of the study.

A magnehelic pressure gauge (0 - 100 mm water gauge) was connected with each chamber by a nylon tube. This was mounted on the trolley and was used to monitor chamber internal pressure.

Extraction of the chambers was accomplished by means of a single fan mounted on the outside wall of the buiiaing withdrawing air through a manifold to which all chambers were connected. The chamber air was withdrawn through coarse and fine filtration media before being exhausted to atmosphere. Extract flow was adjusted using gate valves mounted in the extract ducting between the chamber and filters. The internal pressure within each chamber was maintained at 10 mm water gauge below ambient when operational.
A separate exposure chamber was used for each group of rats. The air control animals were exposed using a similar system to that used for the test group.

- Method of holding animals in test chamber: Animals were not held in

- Source and rate of air: Air entered the chamber through the inlet duct.

- Method of conditioning air: Not specified

- System of generating particulates/aerosols: The vapour of the test substance was produced by metering the liquid from a bulk reservoir pressurised with nitrogen to a copper coil in which vaporisation took place. Dried, filtered air was mixed with the vapour and the resulting vapour/air mixture passed into the inlet air ducting to the chamber. The air to the vaporisation system was heated by passage through a copper coil immersed in a water bath set to a maximum of 70°C. Different concentrations of vapour were produced by using different feed rates of the test liquid.

- Temperature, humidity, pressure in air chamber: The internal pressure of the chambers is adjusted to 1 - 10 mm WG below ambient. The chamber internal pressure relative to ambient was monitored continuously by magnehelic pressure gauges and recorded at 30-minute intervals throughout each exposure. The wet and dry bulb temperatures of a thermohygrometer positioned in each chamber were recorded at 30-minute intervals throughout each exposure. The chamber relative humidity was calculated from these data. The exposure means chamber temperature were 23.7, 23.6, 23.9 and 22.7°C for control, low dose, intermediate group and high dose group, respectively. The exposure means chamber relative humidity were 33, 28, 25and 37% for control, low dose, intermediate group and high dose group, respectively.

- Air flow rate: The total chamber airflow was 150 Iitres/minute, consisting of the vapouriser air supply (25 litres/minute) and diluent air supply of 125 litres/minute. Air flows were measured by tapered tube flow meters situated at the front of a purposebuilt stainless steel trolley on which was mounted the generation apparatus.

The air flow into each chamber was monitored continuously using tapered tube flow meters and recorded at approximately 30-minute intervals throughout each exposure.

- Air change rate:
- Method of particle size determination:
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used: Samples of the test atmosphere of chamber were collected with a syringe to control methylal concentration. Samples were analysed with Gas Chromatography-Flame Ionisation Detection (GC-FID). No potential source of error examined was expected to
exceed 5% of the true value of a quantitative analysis.

- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test atmosphere of chamber were collected with a syringe to control methylal concentration. Samples were analysed with Gas Chromatography-Flame Ionisation Detection (GC-FID). No potential source of error examined was expected to
exceed 5% of the true value of a quantitative analysis.
Details on mating procedure:
Sexually mature Specific Pathogen Free female rats (Crt: CD® BR VAF/Plus strain) which were time-mated to identified males of the same strain, were obtained from Charles River UK Limited, Manston Road, Margate, Kent.

- Impregnation procedure: [artificial insemination / purchased timed pregnant / cohoused] Not specified
- If cohoused: Not specified
- M/F ratio per cage: Not specified
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. Not specified
- Further matings after two unsuccessful attempts: [no / yes (explain)] Not specified
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
- Any other deviations from standard protocol: Not specified
Duration of treatment / exposure:
6-12 days pc
Frequency of treatment:
6-hour daily
Duration of test:
The test duration is 20 days (from Day 0 of presumed pregnancy to Day 20 during which females are killed and subjected to post-mortem observations).
Remarks:
Doses / Concentrations:
386 ppm
Basis:
other: Mean analysed concentrations
Remarks:
Doses / Concentrations:
1954 ppm
Basis:
other: Mean analysed concentrations
Remarks:
Doses / Concentrations:
10068 ppm
Basis:
other: Mean analysed concentrations
No. of animals per sex per dose:
25
Control animals:
yes
Details on study design:
- Dose selection rationale: A range finding test concluded that the exposure levels of 400, 2000, 10000 ppm would be appropriate dosages for an embryotoxicity study in the rat. (see "Cross reference to other study")
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were regularly handled and observed daily for obvious changes or signs of reaction to treatment.
During exposure, clinical signs were recorded as a group sign every 30 minutes, however not all animals were visible each day, therefore clinical signs for individual animals have not been presented during this time.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on Days 2, 6, 8, 10, 12, 14, 16, 18, and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes / No / No data
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
During exposure periods, animals did not have access to food and therefore, food consumption was measured as intake recorded in home cages. Food consumption was measured in cage means value during 8 periods on days 2-5, 6-7, 8-9, 10-11, 12-13, 14-15, 16-17 and 18-19.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations: Daily visual inspection of the water bottles in the home cages indicated a possible effect on water consumption and daily measurement of water consumption was started on day 10, 9 or 8 post coitum according to batch of animals.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: diaphragm and kidney

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Number of live young, litter weight, mean foetal weight, sex ratio of foetus
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
Statistics:
Significance tests, employing analysis of variance followed by an intergroup comparison with the control, were performed on the following parameters:

Bodyweight change, food consumption
Dependent on the heterogeneity of variance between treatment groups, parametric tests (analysis of variance (Snedecor and Cochran 1967) followed by Williams' test (Williams 1971/2) or nonparametric tests (Kruskal-Wallis (Hollander and Wolfe 1973) followed by Shirley's test (Shirley 1977) were used to analyse these data, as appropriate.
All significant (ie < or = 0.05) intergroup differences from the control are reported only where supported by a significant analysis of variance < or = 0.05).

Litter data
For litter data and foetal changes the basic sample unit was the litter and, due to the preponderance of non-normal distributions, non-parametric analyses were routinely used.
Analysis of the incidence and distribution within litters of pre-implantation loss, of in utero deaths and of foetal malformations and anomalies were performed using the Linear by Linear Association test (Agresti, 1990; Agresti, Metha & Patel, 1990), in a step down fashion. Where the Linear by
Linear Association test was not significant (p>0.05), the Kruskal-Wallis test was used to detect nonlinear responses. If the Kruskal-Wallis test was significant < or = 0.01) then pairwise permutation tests (Gibbons, 1985) were used to compare each dose level with the control.

Analysis of mean values for corpora lutea, implantations, litter size (live young), sex ratio, litter weight, foetal- weight and gravid uterine weight were performed using Kruskal- Wallis followed by Shirley's test.
Where 75% or more of the values for a given variable were the same, a Fisher's exact test (Fisher, 1950) was used, when considered necessary.
Indices:
Pre-implantation loss was calculated as (Number of corpora lutea) - (number of implantations).
The total number of embryonic deaths was calculated as (Number of implantations) - (number of live young)
Historical control data:
No
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During exposure, half-closed eyes, reduced response to 'knocking' on the chamber, exaggerated respiratory movements and rubbing of the chin on the grid floor of the exposure basket were noted in High dose group rats. No clinical signs were noted during exposure in the Intermediate and Low dose groups.
At other times, signs noted in High dose group rats included: unsteady gait, lachrymation, salivation, lethargy, half-closed eyes, matted fur on abdomen and wet fur around the urogenital region.
No treatment-related clinical signs were noted in the Intermediate and Low dose groups.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the High dose group, there was a clear reduction in mean bodyweight gain between Days 6 and 12 post coitum compared with controls. From Day 12 onwards, mean weight gain was essentially similar to or higher than that of controls.
No treatment-related effects on the pattern of bodyweight gain were observed in the Intermediate and Low dose groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the High dose group, lower food consumption was recorded during Days 6 to 16 post coitum compared to concurrent controls. Thereafter, food intake was slightly higher than in controls.
No treatment-related effects on food consumption were observed in the Intermediate and Low dose groups.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
In the High dose group, higher water consumption was noted from visual inspection of the water bottles and formal recording of water consumption was started on Day 8 post coitum (Batch A).
Water consumption was markedly higher for High dose group rats compared with concurrent controls.
No treatment-related effects on water consumption were observed in the Intermediate or Low dose group.
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 treatment-related macroscopic changes were noted at post mortem examination of animals at any dosage.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Water consumption
In the High dose group, higher water consumption was noted from visual inspection of the water bottles and formal recording of water consumption was started on Day 8 post coitum (Batch A).
Water consumption was markedly higher for High dose group rats compared with concurrent controls.
No treatment-related effects on water consumption were observed in the Intermediate or Low dose group.
Number of abortions:
no effects observed
Description (incidence and severity):
There were no instances of total litter loss in any group.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
Pre-implantation losses and the implantation rate (events determined prior to the start of treatment) were comparable for all groups.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
No total litter losses observed
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no treatment-related effects upon the incidence and distribution of embryofoetal deaths
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no treatment-related effects upon the incidence and distribution of embryofoetal deaths
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:
no effects observed
Description (incidence and severity):
A total of 24, 23, 25 and 25 dams had live young at Day 20 in Groups 1 to 4 respectively.
Other effects:
not examined
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality
No deaths were observed during this test at any dose level..

Clinical signs
During exposure, half-closed eyes, reduced response to 'knocking' on the chamber, exaggerated respiratory movements and rubbing of the chin on the grid floor of the exposure basket were noted in High dose group rats. No clinical signs were noted during exposure in the Intermediate and Low dose groups.
At other times, signs noted in High dose group rats included: unsteady gait, lachrymation, salivation, lethargy, half-closed eyes, matted fur on abdomen and wet fur around the urogenital region.
No treatment-related clinical signs were noted in the Intermediate and Low dose groups.

Macroscopic pathology
Signs of damaged tail at distal caudal region and red staining fur at left periorbital region were observed in Low dose group rats. Findings of minimal hair loss at fur dorsal thoracic, dorsal lumbar, dorsal cervical and abdominal regions and thinning with protrusion of median liver lobe of diaphragme's central tendinous region, were mentionned on fur of Intermediate dose group rats. No macroscopic pathology was noted among High dose group rats.
No treatment-related macroscopic changes were noted at post mortem examination of animals at any dosage.

Body weight
A reduction was observed in body weight gain between Days 6 and 12 at 10068 ppm.
No treatment-related effects on the pattern of bodyweight gain were observed in the Intermediate and Low dose groups.

Food consumption
A reductioon was observed in food intake between Days 6 and 12 at 10068 ppm. No treatment-related effects on food consumption were observed in the Intermediate and Low dose groups.

Water intake
Water intake was markedly higher than controls during Days 8 to 19 at 10068 ppm. No treatment-related effects on water consumption were observed in the Intermediate or Low dose group.
Dose descriptor:
NOEL
Effect level:
ca. 1 954 ppm (analytical)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
other: maternal toxicity
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Litter weight and mean foetal weight were similar in Control and test groups.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Litter weight and mean foetal weight were similar in Control and test groups.
Description (incidence and severity):
There were no treatment-related effects upon the incidence and distribution of embryofoetal deaths
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No treatment-related effects on sex ratio as assessed by % males/litter were evident in any group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter weight and mean foetal weight were similar in Control and test groups.
Changes in postnatal survival:
not examined
External malformations:
not examined
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 10000 ppm methylal, there was a slight increase in the incidence of skeletal anomalies, principally bipartite and incomplete ossification of thoracic vertebral centra. The findings were considered to be unrelated to treatment with methylal.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
A slight increase was observed in the incidence of dilated ureter in the visceral examination. The findings were considered to be unrelated to treatment with methylal.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter parameters
A total of 24, 23, 25 and 25 dams had live young at Day 20 in Groups 1 to 4 respectively. There were no instances of total litter loss in any group. Pre-implantation losses and the implantation rate (events determined prior to the start of treatment) were comparable for all groups. There were no treatment-related effects upon the incidence and distribution of embryofoetal deaths. Litter weight and mean foetal weight were similar in Control and test groups. No treatment-related effects on sex ratio as assessed by % males/litter were evident in any group.

Malformations
The incidence of malfonnations was considered to be unrelated to treatment with methylal.

Skeletal and visceral anomalies
At 10000 ppm methylal, there was a slight increase in the incidence of skeletal anomalies, principally bipartite and incomplete ossification of thoracic vertebral centra. Also, a slight increase was observed in the incidence of dilated ureter in the visceral examination. The findings were considered to be unrelated to treatment with methylal.

Skeletal variants
The incidence of skeletal variants was considered to be unrelated to treatment with methylal.
Dose descriptor:
NOEL
Effect level:
10 068 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: changes were not considered to be related to exposure to methylal
Abnormalities:
not specified
Developmental effects observed:
no
Treatment related:
no
Relation to maternal toxicity:
not specified
Dose response relationship:
no
Relevant for humans:
not specified
Conclusions:
Exposure of the parent female from Days 6 to 15 of pregnancy to 10068 ppm methylal has no effect on embryofoetal development.
Executive summary:

Twenty-five sexually mature (8-10 weeks old) Specific Pathogen Free female rats (Crt: CD® BR VAF/Plus strain) which were time-mated to identified males of the same strain and supplied by Charles River UK Limited (Manston Road, Margate, Kent.Charles UK) were subjected by whole-body exposure, to the vapour of methylal. Exposure concentrations of 0 (Control), 386, 1954 and 10068 ppm were administered for six hours each day from Days 6 to 15 post coitum inclusive. On Day 20 post coitum, all rats were killed, subjected to post mortem examination, litter values were determined and the foetuses were examined.

Treatment-related effects were confined to the parent females exposed to 10068ppm and comprised lower bodyweight gain with a concomitant reduction in food intake and a markedly greater water intake. No treatment-related effects on the litters were observed.

Foetal pathological examination revealed no changes that were considered to be attributable to exposure to methylal.

The no effect exposure level for this study was 1954 ppm. However, it is concluded that exposure of the parent female from Days 6 to 15 of pregnancy to 10068 ppm methylal has no effect on embryofoetal development.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 February 2017 to 30 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1701099998R
- Expiration date of the lot/batch: 09 January 2020
- Purity test date: 09 January 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Under nitrogen, refrigerated (2 to 8C) and protected from light.
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Readily soluble, stable for up to 4 hours at ambient temperature


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel: Test substance dissolved in water for formulation at concentrations of 20-200 mg/mL
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Reputable commercial supplier
- Age at study initiation: 21-25 weeks
- Weight at study initiation: 2.68 - 4.59 kg
- Fasting period before study: no
- Housing: Suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week. Animals housed in a partial barrier
- Diet (e.g. ad libitum): Teklad 2930 Diet. availability restricted (initially 150 g/animal/day during acclimatization up to 13 days prior to the onset of mating and 200 g/animal/day thereafter).
- Water (e.g. ad libitum): Potable water from public supply available ad libitum
- Acclimation period: 2-3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21°C
- Humidity (%): 45-70%
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 14 hours light : 10 hours dark

IN-LIFE DATES: From: 3 July 2017 To: 4 August 2017
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle:
- Amount of vehicle (if gavage): 5mL/kg
- Lot/batch no. (if required):
- Purity:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Vehicle = water for formulation
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): 1701099998R
- Purity: 99.9958%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity
Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 2 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.

Achieved concentration
Samples of each formulation prepared for administration during the first and last week of treatment were analyzed for achieved concentration of the test item.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: until mating confirmed (1 day)
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. - Not applicable
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: mating observed to take place (all but one animal mated twice) taken as proof of pregnancy. This is considered to be day 0 of the study
- Any other deviations from standard protocol: None
Duration of treatment / exposure:
Days 6 to 28 (inclusive)
Frequency of treatment:
once daily at approximately the same time each day
Duration of test:
Surviving animals killed on day 29 after mating. Females that exhibited pregnancy loss were killed on the day of detection.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 female rabbits per dose level
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels of 100, 300 and 1000 mg/kg/day were selected in conjunction with the Sponsor and were based on the results of a combined pilot and preliminary embryo fetal toxicity study in the New Zealand White Rabbit (Envigo Study Number: HQ40XL).
- Rationale for animal assignment (if not random): Females mating on any one day were evenly distributed amongst the groups. Allocation was controlled to prevent any stock male from providing more than one mated female in each treated group and to prevent more than one sibling female in each group, where possible.
- Other:
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for evidence of ill-health
- Daily cage side observations were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least twice daily for evidence of ill-health or reaction to treatment

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded weekly during acclimatization and on Days 0, 3, and 6-29 after mating

FOOD CONSUMPTION : Yes
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 1 after mating.


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: Detailed necropsy with full macroscopic examination of all tissues performed. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

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:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
Statistics:
The following data types were analyzed at each timepoint separately:
Body weight,
Gravid uterine weight and adjusted body weight
Food consumption
Litter size and survival indices
Fetal, placental and litter weight

The following comparisons were performed:
Group 1 vs 2, 3 and 4

The following sequence of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, live young, fetal, placental and litter weight data: Bartlett's test for variance homogeneity, a parametric monotonic trend test, Dunnett's test, a variety of non-parametric tests

For litter size and survival indices and fetal, placental and litter weight and gravid uterine weight data, if 75% of the data (across all groups) were the same value, for example c, Fisher's exact tests were performed.

Pre/post implantation loss and sex ratio were analyzed by generalized mixed linear model with binomial errors, a logit link function and litter as a random effect.
For resorptions, each treated group was compared to control by exact Wilcoxon rank sum test.
Historical control data:
Historical control data was taken from OECD 414 studies performed on the same species at Envigo during June 2012 to March 2017
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
At routine physical examination there were a slightly higher number of females at 1000 mg/kg/day that showed reduced faecal output.
No signs were observed in association with dose administration that were considered to relate to administration of Methylal.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One Group 1 (Control) female was found dead on GD14, death was attributed to accidental trauma during dose administration. One Group 2 (100 mg/kg/day) female was found dead on GD19 , the cause of death was unclear. Both these rabbits were not pregnant. One Group 4 (1000 mg/kg/day) female was killed prematurely with evidence of abortion on GD27, macroscopic examination revealed fetal material in the stomach and pale liver.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
When compared with Controls, females receiving 1000 mg/kg/day showed slightly but significantly greater weight loss during the first two days of treatment (GD6-8; p<0.01); thereafter body weight gain was similar to Controls (GD8-29). Body weight change at 100 or 300 mg/kg/day was unaffected by treatment.
Maternal weight change following adjustment for the gravid uterine weight showed no adverse effect of treatment at dose levels up to and including 1000 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
From GD6 to GD16 females receiving 1000 mg/kg/day showed statistically significantly low food consumption when compared with the Controls; thereafter consumption was similar to Controls. Food consumption at 100 or 300 mg/kg/day was considered to be unaffected by treatment.
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:
no effects observed
Description (incidence and severity):
There were no macroscopic findings on GD29 that could be attributed to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Description (incidence and severity):
As there were no macroscopic changes in any maternal organs/tissues examined, histopathology was not performed.
Histopathological findings: neoplastic:
not examined
Number of abortions:
effects observed, non-treatment-related
Description (incidence and severity):
One female from Group 4 (1000 mg/kg/day) was killed with evidence of abortion on GD27; uterine examination revealed 10 empty implantation sites
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
One doe in the 1000 mg/kg/day group aborted her litter on GD27. Among the surviving does in the 1000 mg/kg/day dose group, the mean numbers of resorptions and resultant post implantation loss were significantly increased compared to Controls (p<0.05); further, the percentage of post implantation loss was outside the historical control data (HCD) range (3.5-15.8% post implantation loss, 32 main embryo-fetal studies, 2012 to 2017). In the mid dose (300 mg/kg/day) group, the reported percentage of postimplantation loss exceeded that of the Historical Control Database; however, the mean number of resorptions was not statistically different from concurrent controls and it fell within the historical control range (0.4-1.3 resorption, 32 main embryo-fetal studies, 2012 to 2017). Further, the increased post-implantation loss at 300 mg/kg/day appears to be driven by data for a single doe that showed 75% post-implantation litter loss. When the data for this doe are excluded, the mean post-implantation loss at 300 mg/kg/day (12.9%) is less than that of the low dose group.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
At 300 and 1000 mg/kg/day, the mean number of resorptions and resultant post implantation loss were slightly high when compared with Controls (p<0.05 at 1000 mg/kg/day) and the historical control data range (3.5-15.8% post implantation loss, 32 main embryo-fetal studies, 2012 to 2017). However, review of the individual data shows that the difference at 300 mg/kg/day can be attributed to a single female/litter with 75% post-implantation loss. When the data for this litter is excluded, the mean post-implantation loss at 300 mg/kg/day (12.9%) is less than that of the low dose group.
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
At 100 and 1000 mg/kg/day, the late resorptions were above the historical control data (0.0-0.4 late resorption, 32 main embryo-fetal studies, 2012 to 2017). However, the difference with the Controls was not statistically significant. Furthermore, there is no effect on overall litter size at dose levels up to and including 1000 mg/kg/day.
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
pre and post implantation loss
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
not examined
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In all groups that received Methylal there were a relatively small number of major abnormalities; the majority were unrelated to each other and considered spontaneous malformations.
At 100, 300 and 1000 mg/kg/day, disparate cranial abnormalities (Acephaly, Anencephaly and Cranioschisis) occurred in single fetuses, which also exhibited related eye/palate, cervical vertebral and limb abnormalities.
When compared with the concurrent control and historical control data range there was an increased incidence of full supernumerary 13th rib, 20 thoracolumbar vertebrae, unilateral shift of pelvic girdle and a decrease in incidence of 7th costal cartilage not connected to sternum at 1000 mg/kg/day (10-48 full supernumerary 13th rib, 1-35 20 thoracolumbar vertebrae, 1-9 unilateral shift of pelvic girdle and 3-22 7th costal cartilage not connected to sternum, 10 main embryo-fetal studies, 2016 to 2017). Nevertheless, these are common anatomical variations observed in rabbits.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
In all groups that received Methylal there were a relatively small number of major abnormalities; the majority were unrelated to each other and considered spontaneous malformations.
Many of the other malformations observed in these fetuses were a consequence of the cranial defect. These included the malformations of the eyes, orbits, palate, facial bones and tympanic annula
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased post-implantation loss
Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Treatment related:
yes

Number of pregnant and non-pregnant dams:

 

Control

0 mg/kg/day

Methylal

100 mg/kg/day

Methylal

300 mg/kg/day

Methylal

1000 mg/kg/day

Number of pregnant dams

19

19

18

20

Number of non-pregnant dams

3

3

4

2

Number of dams with abortions, early deliveries, stillbirths, resorptions, and/or dead fetuses:

Number of dams with

Control

0 mg/kg/day (N = 19)

Methylal

100 mg/kg/day (N = 19)

Methylal

300 mg/kg/day (N = 18)

Methylal

1000 mg/kg/day (N = 20)

Abortions

0

0

0

1

Early deliveries

0

0

0

0

Stillbirths

0

0

0

0

Resorptions

9

10

12

13

Dead fetuses

0

0

0

0

Pre-and post-implantation loss, number and percent:

 

Control

0 mg/kg/day

Methylal

100 mg/kg/day

Methylal

300 mg/kg/day

Methylal

1000 mg/kg/day

Pre-implantation loss (Mean number)

Mean

2.7

1.4

1.8

2.6

N

19

19

18

19

Post-implantation loss (Mean number)

Mean

0.6

1.3

1.11

1.5

N

19

19

18

19

Pre-implantation loss (%)

Mean

20.2

13.9

19.7

18.8

N

19

19

18

19

Post-implantation loss (%)

Mean

9.2

13.2

16.3

16.9

N

19

19

18

19

Body weight, body weight change and gravid uterine weight:

 

Control

0 mg/kg/day

Methylal

100 mg/kg/day

Methylal

300 mg/kg/day

Methylal

1000 mg/kg/day

Body weight on day 29 (kg)

Mean

3.8

3.75

3.66

3.72

SD

0.443

0.267

0.295

0.431

N

19

19

18

19

Body weight change D6-29 (kg)

Mean

0.26

0.29

0.23

0.19

SD

0.146

0.131

0.173

0.213

N

19

19

18

19

Gravid uterine weight (kg)

Mean

0.49

0.47

0.41

0.47

SD

0.147

0.074

0.157

0.124

N

19

19

18

19

Mean number of live offspring:

Mean number of live offspring

Control

0 mg/kg/day

Methylal

100 mg/kg/day

Methylal

300 mg/kg/day

Methylal

1000 mg/kg/day

Mean

7.9

7.5

6.5

7.4

SD

2.58

1.71

2.94

2.19

N

19

19

18

19

 

Percent of live offspring*:

Live offspring compared to

Control

0 mg/kg/day

Methylal

100 mg/kg/day

Methylal

300 mg/kg/day

Methylal

1000 mg/kg/day

Concurrent controls (%)

100

94.94

82.28

93.67

Number of implantations (%)

92.94

85.23

85.53

83.15

*No dead foetuses were observed

Number of corpora lutea:

 

Control

0 mg/kg/day

Methylal

100 mg/kg/day

Methylal

300 mg/kg/day

Methylal

1000 mg/kg/day

Mean

10.5

10.1

9.3

10.9

SD

1.26

1.88

2.05

1.54

N

19

19

18

19

 

Mean foetal/pup body weight by sex and sexes combined:

 

Control

0 mg/kg/day

Methylal

100 mg/kg/day

Methylal

300 mg/kg/day

Methylal

1000 mg/kg/day

Male fetal weight (g)

Mean

42.2

40.9

42.1

39.6

SD

4.56

5.98

4.8

5.36

N

19

19

16

19

Female fetal weight (g)

Mean

40.0

39.9

42.2

40.0

SD

4.00

4.01

6.08

4.55

N

17

19

17

19

Overall fetal weight (g)

Mean

41.5

40.3

42.8

39.6

SD

4.23

4.12

5.68

4.02

N

19

19

18

19

Fetal examinations - major abnormality findings – group percentage incidences (part 1)

Group

:

 1

 2

 3

 4

Compound

:

Control

Methylal

Methylal

Methylal

Dose (mg/kg/day)

:

0

100

300

1000

 

 

Fetuses

 

 

Litters

 

Group

 

1

2

3

4

 

 

1

2

3

4

 

Number Examined

 

150

142

117

141

 

 

19

19

18

19

 

Total Percentage Affected

Total Number Affected

 

2.7

4

2.1

3

1.7

2

2.8

4

 

 

10.5

2

15.8

3

11.1

2

15.8

3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Head

 

 

 

 

 

 

 

 

 

 

 

Skeletal                                     $

Bent/misshapen mandibles

0.0

0.0

0.0

1.5

 

 

0.0

0.0

0.0

5.3

 

$

Displaced tympanic annula

0.0

0.0

0.0

1.5

 

 

0.0

0.0

0.0

5.3

 

$

Anencephaly

0.0

0.0

0.0

1.5

 

 

0.0

0.0

0.0

5.3

 

$

Small/misshapen nasals

0.0

1.5

0.0

0.0

 

 

0.0

5.3

0.0

0.0

 

$

Flattened cranium/protrusion supraoccipital

1.4

0.0

0.0

0.0

 

 

5.3

0.0

0.0

0.0

 

Visceral                                       

Absent eye

0.0

0.0

0.0

0.7

 

 

0.0

0.0

0.0

5.3

 

External

Acephaly

0.0

0.7

0.0

0.0

 

 

0.0

5.3

0.0

0.0

 

 

Anencephaly

0.0

0.0

0.9

0.0

 

 

0.0

0.0

5.6

0.0

 

 

Cranioschisis

0.0

0.7

0.0

0.0

 

 

0.0

5.3

0.0

0.0

 

 

Open eye lid(s)

0.0

0.7

0.0

0.0

 

 

0.0

5.3

0.0

0.0

 

 

Protruding eye(s)

0.0

0.0

0.9

0.0

 

 

0.0

0.0

5.6

0.0

 

 

Cleft palate

0.0

0.7

0.0

0.0

 

 

0.0

5.3

0.0

0.0

 

 

Protruding tongue

0.0

0.0

0.9

0.0

 

 

0.0

0.0

5.6

0.0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Note: Individual fetuses/litters may occur in more than one category.

$ Findings noted at skeletal examination, therefore % was calculated from number of intact skeletons

 

Fetal examinations - major abnormality findings - group percentage incidences (part 2)

Group

:

 1

 2

 3

 4

Compound

:

Control

Methylal

Methylal

Methylal

Dose (mg/kg/day)

:

0

100

300

1000

 

 

Fetuses

 

 

Litters

 

Group

 

1

2

3

4

 

 

1

2

3

4

 

Number Examined

 

150

142

117

141

 

 

19

19

18

19

 

Total Percentage Affected

Total Number Affected

 

2.7

4

2.1

3

1.7

2

2.8

4

 

 

10.5

2

15.8

3

11.1

2

15.8

3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Cervical/Thoracic

 

 

 

 

 

 

 

 

 

 

 

Skeletal

Thoracic kyphosis

0.0

0.7

0.0

0.0

 

 

0.0

5.3

0.0

0.0

 

 

Cervical lordosis

0.0

0.7

0.0

0.0

 

 

0.0

5.3

0.0

0.0

 

 

6 cervical vertebra present

0.0

0.0

0.0

0.7

 

 

0.0

0.0

0.0

5.3

 

 

Misshapen/disorganised cervical vertebra

0.0

0.7

0.0

0.0

 

 

0.0

5.3

0.0

0.0

 

 

Cervical spina bifida

0.0

0.7

0.0

0.0

 

 

0.0

5.3

0.0

0.0

 

 

Thoracic scoliosis

0.0

0.7

0.0

0.0

 

 

0.0

5.3

0.0

0.0

 

 

Multiple thoracic vertebral/rib abnormalities

0.0

0.7

0.0

0.0

 

 

0.0

5.3

0.0

0.0

 

 

Interrupted thoracic to caudal vertebra

0.0

0.0

0.0

0.7

 

 

0.0

0.0

0.0

5.3

 

 

Dorsoventral distortion of sternum

0.0

0.7

0.0

0.0

 

 

0.0

5.3

0.0

0.0

 

 

5 normal pairs of ribs present

0.0

0.0

0.0

0.7

 

 

0.0

0.0

0.0

5.3

 

Visceral

Dilated ascending aorta/aortic arch

0.7

0.0

0.0

1.4

 

 

5.3

0.0

0.0

10.5

 

 

Narrow pulmonary trunk

0.7

0.0

0.0

0.7

 

 

5.3

0.0

0.0

5.3

 

 

Dorsally displaced pulmonary trunk

0.0

0.0

0.0

0.7

 

 

0.0

0.0

0.0

5.3

 

 

Transposition of ascending aorta with pulmonary trunk

0.0

0.0

0.0

0.7

 

 

0.0

0.0

0.0

5.3

 

 

Membranous ventricular septal defect

0.7

0.0

0.0

0.7

 

 

5.3

0.0

0.0

5.3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Note: Individual fetuses/litters may occur in more than one category.

 

Fetal examinations - major abnormality findings - group percentage incidences (part 3)

Group

:

 1

 2

 3

 4

Compound

:

Control

Methylal

Methylal

Methylal

Dose (mg/kg/day)

:

0

100

300

1000

 

 

Fetuses

 

 

Litters

 

Group

 

1

2

3

4

 

 

1

2

3

4

 

Number Examined

 

150

142

117

141

 

 

19

19

18

19

 

Total Percentage Affected

Total Number Affected

 

2.7

4

2.1

3

1.7

2

2.8

4

 

 

10.5

2

15.8

3

11.1

2

15.8

3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lumbar (and abdominal)/Sacral/Caudal

 

 

 

 

 

 

 

 

 

 

 

Skeletal

Lumbar kyphosis

0.7

0.0

0.0

0.0

 

 

5.3

0.0

0.0

0.0

 

 

Fused/misshapen disorganised caudal vertebra

1.3

0.0

0.0

0.0

 

 

5.3

0.0

0.0

0.0

 

 

Brachyury

2.0

0.0

0.0

0.0

 

 

5.3

0.0

0.0

0.0

 

External

Holorachischisis

0.0

0.0

0.9

0.0

 

 

0.0

0.0

5.6

0.0

 

 

Gastroschisis

0.0

0.0

0.9

0.0

 

 

0.0

0.0

5.6

0.0

 

 

Omphalocele

0.0

0.0

0.9

0.0

 

 

0.0

0.0

5.6

0.0

 

 

Lumbar/sacral spina bifida

0.7

0.0

0.0

0.0

 

 

5.3

0.0

0.0

0.0

 

 

Tail threadlike

1.3

0.0

0.0

0.0

 

 

5.3

0.0

0.0

0.0

 

Visceral

Absent adrenal(s)

0.0

0.0

0.0

0.7

 

 

0.0

0.0

0.0

5.3

 

 

Displaced ovarie(s)

0.0

0.0

0.0

0.7

 

 

0.0

0.0

0.0

5.3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Appendicular

 

 

 

 

 

 

 

 

 

 

 

Skeletal

Bent clavicle

0.0

0.7

0.0

0.0

 

 

0.0

5.3

0.0

0.0

 

 

Ectrodactyly hindpaw(s)

0.0

0.0

0.9

0.0

 

 

0.0

0.0

5.6

0.0

 

 

Brachydactyly hindpaw(s)

0.0

0.7

0.9

0.0

 

 

0.0

5.3

5.6

0.0

 

External

Malrotated hindlimb(s)

0.0

0.7

0.9

0.0

 

 

0.0

5.3

5.6

0.0

 

 

Unable to straighten fore/hindlimb(s)

0.7

0.0

0.9

0.7

 

 

5.3

0.0

5.6

5.3

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Note: Individual fetuses/litters may occur in more than one category.

Conclusions:
Based on the body weight loss, low food consumption and increased post-implantation loss at 1000 mg/kg/day, the intermediate dose level of 300 mg/kg/day is considered to be both the maternal and fetal no observed adverse effect level (NOAEL).
Executive summary:

An OECD 414 study was performed to assess the influence of Methylal, a solvent, on pregnant animals and on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of the New Zealand White rabbit.

Three groups of 22 females received Methylal at doses of 100, 300 or 1000 mg/kg/day by oral administration, from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle, water for formulation, at the same volume dose as the treated groups. Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Three animals died prior to scheduled termination:

Group 1 female no 11 was found dead on GD14; death attributed to accidental trauma during dose administration.

Group 2 female no 31 was found dead on GD19. Terminal signs included reduced food consumption but clinical condition appeared normal. There were no macroscopic findings at necropsy; the cause of death remains unclear.

Group 4 female no 86 was killed prematurely with evidence of abortion on GD27. Terminal signs included reduced food consumption, slight weight loss and thin build with pink/red staining in the cage and on the cage tray paper. Macroscopic examination revealed fetal material in the stomach and a pale liver.

At routine physical examination there were a slightly higher number of females at 1000 mg/kg/day that showed reduced faecal output.

Females receiving 1000 mg/kg/day showed slightly but significantly greater weight loss during the first two days of treatment (GD6-8; p<0.01). From GD6 to GD16 females receiving 1000 mg/kg/day showed statistically significantly low food consumption when compared with the Controls; thereafter consumption was similar to Controls

Bodyweight gain and food consumption at 100 and 300 mg/kg/day were unaffected by treatment and the maternal weight change following adjustment for the gravid uterine weight showed no adverse effect of treatment at dose levels up to and including 1000 mg/kg/day.

There were no macroscopic findings on GD29 that could be attributed to treatment.

At 300 and 1000 mg/kg/day the mean number of resorptions and resultant post-implantation loss was slightly high when compared with Controls; with the difference attaining statistical significance at 1000 mg/kg/day (p<0.05). However, review of the individual data shows that this difference at 300 mg/kg/day can be attributed to a single female/litter with 75% post-implantation litter loss; when the data for this litter is excluded, the mean post-implantation loss is less than that of the low dose group. There was no adverse effect on the live litter size, mean fetal or placental weight.

Examination of fetuses at 100, 300 and 1000 mg/kg/day revealed low incidences of major cranial abnormalities (Acephaly, Anencephaly and Cranioschisis) with related eye/palate, cervical vertebral and limb abnormalities. However, the observed cranial malformations are located in separate anatomic locations, undergo disparate morphogenetic processes, and occur at different times in gestation. Therefore, they are not considered related findings.

At 1000 mg/kg/day, fetal skeletal examination revealed an increased incidence of full supernumerary 13th rib, 20 thoracolumbar vertebrae, unilateral shift of pelvic girdle and a decrease in incidence of 7th costal cartilage not connected to sternum. These conditions are commonly observed skeletal variants in rabbits.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rabbit
Quality of whole database:
2nd species (rabbit - oral route): in vivo prenatal developmental toxicity performed according to OECD 414 and GLP --> Klimisch code 1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
31 814.88 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
1st species (rat - inhalation route): in vivo prenatal developmental toxicity performed according to OECD 414 and GLP --> Klimisch code 1
Other species: QSAR prediction of developmental toxicity to dimethoxymethane using the Leadscope structural dysmorphogenesis in rat, rodent, mouse and rabbit: --> Klimisch code 2
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Testing in a first species

In in vivo Prenatal developmental toxicity test performed on rat by inhalation route according to OECD TG 414 and GLP, effects observed for developmental toxicity were number of corpora lutea, number and distribution of live young and embryofoetal deaths, foetal weight, foetal abnormalities (malformation, skeletal and visceral abnormalities, skeletal variants); sex ratio.

Ppm was converted to mg/m3 with the conversion factor of 1ppm=3.16 mg/m3 presented in Handbook (Verschueren, 2001).

Verschueren K (2001). Handbook of environmental data on organic chemicals, Fourth ed., John Wiley, New York, p. 888-889.

Testing in a second species

Testing in a second species is a requirement according to REACH Regulation in Annex X.

In order to follow the ‘Avoidance of Unnecessary Testing’ stated in Article 25 of Regulation 1907/2006, testing on vertebrate animals for the purposes of this Regulation shall be undertaken only as a last resort.

Robust QSAR modeling based on developmental toxicity on rabbit, rat, rodent and mouse with Leadscope Developmental toxicity Suite is used in order to demonstrate the absence of significance in sensitivity of species tested. The four models show negative predictions for developmental toxicity.

Nevertheless, if the Authorities consider a developmental toxicity test in a second non-rodent species like the rabbit for inevitable, the test should be performed via the oral route and not via the inhalation route for the following reasons:

-         A developmental toxicity test in rabbits via inhalative exposure is neither in line with animal welfare legislation as this test design causes much more stress for the test animals as compared to the standard oral route nor it is scientifically justified for the different reasons described below. In addition, after contacting several CROs it was confirmed that for rabbits in contrast to rats a nose only exposure is not feasible. Thus whole body exposure would be the only possible alternative. Whole body exposure is however very problematic with regard to proper dosing/bioavailable doses and especially pregnant rabbits are known to be very sensitive to external influences/stress.

-         There is only very limited experience with inhalation test in rabbits in the known CROs and practically none with developmental toxicity tests in rabbits using this route of exposure. If at all, only a very limit historical dataset is available which will definitively lead to severe problems when interpreting the test results.

-         The aim of this study is to investigate the developmental toxicity (hazard profile) of the substance. Developmental toxicity is without doubt a systemic effect which is independent of the route of exposure, as long as the substance is considered to become well bioavailable by the route of exposure used. In the available acute toxicity studies, the test substance was shown to be absolutely non-toxic independent of the route of exposure with very high LD50/LC50 values of 6423 mg/kg bw/day (oral) and 68690 mg/m3(inhalation). Thus, there is no indication that inhalation route is of specific or higher sensitivity than the oral route.

-         OECD 414 testing in rats via the inhalation route did not reveal any adverse effects with regard to developmental toxicity up to a limit concentration of 10068 ppm (measured). Thus it was proven that inhalation exposure does not lead to adverse effects on development. As outlined above, developmental toxicity is a systemic effect which is independent from the route of exposure. Thus, testing the second species via the oral route (most likely again up to the limit dose) is completely sufficient.

-         There are not indications that the toxikokinetic behavior of the substance differ between the oral and the inhalation route.

 

Thus, if such a test is really unavoidable, the test should be done via the oral route to obtain robust results and being in line with the animal welfare legislation.

Justification for selection of Effect on developmental toxicity: via inhalation route:

Selected as Klimisch code 1 (OECD 414 and GLP) data.

Justification for classification or non-classification

No Classification for developmental toxicity is proposed. Methylal is not considered to have a specific intrinsic property to affect reproduction/development.

Additional information