Dimethoxymethane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 1996-January 1997

Reliability:

1 (reliable without restriction)

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Methylal

IUPAC Name:

Methylal

Details on test material:

- Name of test material (as cited in study report): Methylal
- Substance type: Solvent
- Physical state: Colourless liquid
- Analytical purity: 99.5%
- Lot/batch No.: 951206
- Stability under test conditions: responsability of the sponsor
- Storage condition of test material: Ambient temperature
- Other: Data received: 1996-08-07

Test animals

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

Administration / exposure

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).

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
386 ppm
Basis:
other: Mean analysed concentrations

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1954 ppm
Basis:
other: Mean analysed concentrations

Doses / concentrations 3

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")

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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.

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

no

Treatment related:

no

Relation to maternal toxicity:

not specified

Dose response relationship:

no

Relevant for humans:

not specified

Applicant's summary and conclusion

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.