N,N-dimethylacetamide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

INHALATION: No effects on reproductive performance observed in an inhalation one-generation reproduction toxicity study in rats up to the highest tested dose of 300 ppm (1080 mg/m³) (Ferenz & Kennedy, 1986). Furhtermore, no adverse effects on fertility parameters were observed ina male fertility study in rats (Wang, 1989) and in a sperm abnormality test (NIOSH, 1980).

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

MTD not reached [but high dose level was clearly above OEL of 10 ppm]; no histopathology of reproductive organs [but examined in chronic inhalation studies]

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
- Purity: >99.9 %
- Source: DuPont, commercial grade
No further details available.

Species:

rat

Strain:

other: Crl:CD®

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, NY
- Age at study initiation: (P) 35 days old (arrival: 22 days old)
- Acclimatisation: 13 days
- Weight at study initiation: no data
- Housing: singly
- Certified diet and water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 72-76 °F
- Humidity: 40-60 %
- Air changes: 12-15 changes per hour
- Photoperiod: 12 hours dark/12 hours light

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Remarks:

nitrogen and air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Liquid DMAC was syringe-driven through a preheated (200-225 °C) inclined tube furnace; nitrogen (1-2 L/min) carried vapours through the tube; stream diluted with air (500 L/min), and mixture introduced into the chamber; temperature, oxygen, and humidity in the chamber were measured for each exposure.
Exposure chamber volume: 750-900 L; compartmentalized stainless steel, wire-mesh cage modules.

There were 6 groups of 10 males and 20 females. Group I (0 ppm) was exposed to room air. Both males and females in Groups II - IV (30, 100, and 300 ppm, respectively) were exposed to DMAC. In Group V, only males were exposed (300 ppm, females unexposed) and in Group VI, only females were exposed (300 ppm).

Exposures were 6 hours/day and 5 days/week for 10 weeks (prebreeding), then 7 days/week for 7-8 weeks (breeding, gestation, and lactation). The exposure period started when rats were 35 days old and ended for the males upon completion of the breeding period (after 63 exposures), and for the females at 21 days post partum (after 89-104 exposures). Half of the adult males in each group were sacrificed after the breeding period; the other half after a recovery period of 20 days. Dams were sacrificed on day 21 post partum.

Details on mating procedure:

- M/F ratio per cage: 1/2
- Length of cohabitation: 5 days

When rats were 100 days old, each male was placed in a cage with two females; after each 5 days of cohabitation males were rotated among pairs of females in the same treatment group; each female was paired with three different males; females were checked daily for signs of mating .

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Atmospheric concentrations were analyzed on a Hewlett-Packard 5790A gas chromatograph (GC) with a flame ionization detector; the GC was calibrated daily by injection of standards; vapour concentrations were analyzed at 30- to 60-min intervals by drawing atmosphere through tandem glass impingers.

Duration of treatment / exposure:

Males: 63 exposures
Females: 89-104 exposures

Frequency of treatment:

6 h/day, 5 days/week for 10 weeks (prebreeding), then 7 days/week for 7-8 weeks (breeding, gestation, and lactation)

Details on study schedule:

After exposure on gestation day 19 (GD19) pregnant females were housed singly in delivery boxes with nesting material; they were not exposed from GD 21 through Day 3 postpartum (PP). On Day 4 PP, the number of pups in each litter was reduced to eight (four of each sex where possible) using a table of random numbers; pups remained in the delivery boxes during the dams further exposure periods.

At 21 days of age, liver and gonadal tissue from one male and one female offspring per litter were weighed and stored.

Dose / conc.:

30 ppm (nominal)

Remarks:

31.1 ± 4.86 ppm (analytical);
110 mg/m³

Dose / conc.:

100 ppm (nominal)

Remarks:

101 ± 13.4 ppm (analytical);
360 mg/m³

Dose / conc.:

300 ppm (nominal)

Remarks:

291 ± 29.9 ppm (analytical);
1080 mg/m³

No. of animals per sex per dose:

10 males and 20 females

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Dose selection corresponded to the results of Horn et al. (1961; see Section 7.5.3).

Parental animals: Observations and examinations:

CLINICAL SIGNS:
Daily observations for clinical signs of toxicity were made prior to each exposure.

BODY WEIGHT:
Parental rats were weighed weekly until breeding began; dams were weighed on Days 1, 4, 14, and 21 PP.

OTHER:
Records of mating performance, fertility, gestation length, and lactation performance were maintained.
Mating index, Fertility index, Males impregnating females (%), Gestation length (days), and Gestation index were calculated.

Litter observations:

PARAMETERS EXAMINED
The number of pups and their viability, sex (days 1, 4, 14, 21 PP) and weight (day 1, 4, 21) were determined during lactation; litters were observed daily for clinical signs and the following developmental parameters: pinna detachment, hair growth, and eye opening.

Postmortem examinations (parental animals):

ORGAN WEIGHTS:
After sacrifice, livers and testes were weighed in males, and livers and ovaries were weighed in females.

Postmortem examinations (offspring):

ORGAN WEIGTHS
At 21 days of age, liver and gonadal tissue from one male and one female offspring per litter was weighed.

Statistics:

A one-way analysis of variance was performed (body, liver, and gonad weights).
Mating, fertility, and gestation indices were analyzed by the Fisher's exact test and the Cochran-Armitage test for trend.
All other reproductive parameters were analyzed by Kruskal-Wallis, Jonckheere, and Mann-Whitney U tests.
Significance was judged at the 5 % level.

Reproductive indices:

Fertility index (% matings resulting in pregnancy)
Gestation index (% matings resulting in live births)

Offspring viability indices:

Viability index (% rats born that survived 4 days or more)
Lactation index (% pups alive at 4 days that survived to 21 days PP)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment related clinical signs.

Dermal irritation (if dermal study):

not examined

Mortality:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

During the first 3 weeks of exposure a significant decrease in body weight was measured in males of group V (300 ppm); this effect was not found in males of group IV or in any other test group.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no effects on reproduction indices.

Dose descriptor:

NOAEC

Effect level:

100 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: reduced body weight in males at 300 ppm (1080 mg/m³)

Dose descriptor:

NOAEC

Effect level:

300 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

organ weights and organ / body weight ratios

Dose descriptor:

NOAEC

Effect level:

300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment related clinical signs were observed.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

No effects were seen at any dose group on viability.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of Group VI pups (300 ppm to dams), both male and female, were significantly lower than controls on Days 1 and 21 and when weighed as a group on Day 4. This effect was not observed in Group IV female pups where dams (as well as sires) were exposed to the same concentration of DMAC. Group IV male pups did, however, have significantly lower body weights on Day 21. Since this effect was seen in Group VI male pups on Day 21, it may have been related to dam exposure to 300 ppm DMAC. No other body weight effects were seen.

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Groups IV and VI pups, male and female, showed a significant increase in liver/body weight ratios, but not in absolute liver weights; this increase was due to the fact that final body weights of pups in these groups were lower than those of controls.

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There were no effects on the lactation index or on developmental parameters.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

100 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reduced body weight of offspring at 300 ppm (1080 mg/m³)

Critical effects observed:

no

Reproductive effects observed:

no

Mean weights of sires in group V vs. control weights: 131 vs. 139 g at 3 days, 181 vs. 195 g at 10 days, 233 vs. 249 g at 17 days.

Mean relative liver weight in group IV vs control (± standard deviation): males 4.63 ± 0.76 vs. 3.91 ± 0.343 and females 5.50 ± 0.306 vs 4.76 ± 0.306 (significant in males and females).

Mean pup weights in group VI (300 ppm, only females exposed) vs control weights:

Day 1, males 5.80 vs. 6.17 g, females 5.53 vs. 5.98 g

Day 4, males/females 7.24 vs. 7.81 g

Day 21, males 34.0 vs. 39.2, females 32.9 vs. 38.0 g

Conclusions:

No effects on reproductive parameters in rats were found after repeated inhalation exposure at a dose level of 300 ppm (1080 mg/m³).

Executive summary:

Ferenz and Kennedy (1986) performed a one-generation study equivalent to OECD 415 (unknown GLP compliance) using Crl:CD^®rats. Ten males and twenty females were exposed via inhalation to either 30, 100, or 300 ppm (nominal concentration) of the test substance. Analytical concentrations of the test substance were 31.1± 4.86 ppm, 101.0 ± 13.4 ppm, and 291.0 ± 29.9 ppm with ranges 4.10-65.1 ppm, 27.2-168 ppm, and 91.9-402 ppm, respectively.Animals were exposed to the test substance 6 hours/day, 5 days/week for ten weeks (pre-breeding), then 7 days/week for 7-8 weeks (breeding, gestation, and lactation). Control animals were sham exposed using nitrogen and air. After exposure on gestation day 19 (GD19) pregnant females were housed singly in delivery boxes with nesting material; they were not exposed from GD 21 through Day 3 postpartum (PP). On Day 4 PP, the number of pups in each litter was reduced to eight (four of each sex where possible) using a table of random numbers; pups remained in the delivery boxes during the dam’s further exposure periods. Clinical signs were recorded daily prior to exposure (parental animals). After sacrifice, livers and testes/ovaries were weighed in males and females, respectively. At day 21 post-natal, liver and gonadal tissue from one male and one female pup per litter were weighed. In parental all examined parameters were not affected by treatment except body weight. During the first 3 weeks of exposure a significant decrease in body weight was measured in males of group V (300 ppm); this effect was not found in males of group IV or in any other test group. No effects on reproductive performance related to treatment were identified. The NOAEC for parental animals was set to 100 ppm (nominal) based on the reduction of body weight. Comparable to findings in parental animals, only body weight changes were affected in relation to treatment. Mean body weights of pups (300 ppm to dams), both male and female, were significantly lower than controls. There were no effects on the lactation index or on developmental parameters. The NOAEC for the F1 generation was set to 100 ppm based on the reduced body weight.    

Reference 2

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

only males exposed, dams sacrificed at GD20

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

yes

Remarks:

only males exposed, dams sacrificed at GD20

Principles of method if other than guideline:

The present study was initiated to investigate the potential of DMAC for causing reproductive effects in male rats and to provide some data for human risk estimation.

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
- Purity: 99.8 %
- Impurities: Water, acetic acid, monomethylacetamide, dimethylamine
- Stability: No decomposition during study (measured by IR spectroscopy)
- Source: Monsanto Comp.
No further details available.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Strain: Crl:CD [SD] Br

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Acclimatisation period: 2 weeks
- Certified food and water: ad libitum
- Housing: single

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 2 °F
- Humidity: 40-60 %
- Photoperiod: 12 hours dark/12 hours light

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Remarks on MMAD:

MMAD / GSD: Not applicable (vapour)

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1750 L Rochester-style stainless steel and glass inhalation chambers
- Air change rate: 12 air changes per h

TEST ATMOSPHERE
Vapor atmospheres of DMAC were produced by metering liquid to Laskin-style nebulizers (Drew et al., 1978). Concentrations of DMAC in the inhalation chambers were analyzed with a Wilks MIRAN IA general purpose gas analyzer and recorded four times on each exposure day. Measurements for DMAC were also made on each exposure day in the control chamber.

Details on mating procedure:

Beginning on day 64 of the study (after 43 exposure days), males were initially cohoused with 2 females each. Each morning, observations were made for the presence of copulatory plugs and vaginal smears were taken for determination of the presence of sperm. Copulation was considered confirmed if a copulatory plug was present (for a single female housed with a male) or if sperm were observed in the vaginal smear. The day of confirmed copulation was designated as gestation day 0 and females with confirmed copulation were caged individually. Females in the initial mating groups were cohoused with males (except during exposure) until confirmed copulation nights had occurred without confirmed copulation. Males that did not mate with both initially assigned females and females without confirmed copulation were reassigned into new mating groups (maximum of two females per male) until confirmed copulation occurred or a maximum of five additional mating nights.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of DMAC in the inhalation chambers were analyzed with a Wilks MIRAN IA general purpose gas analyzer and recorded four times on each exposure day. Measurements for DMAC were also made on each exposure day in the control chamber. Nominal concentration measurements were made at least twice daily by measuring air flows and amount of test material delivered to the test chambers.

Duration of treatment / exposure:

total of 69 exposure days (14 weeks)

Frequency of treatment:

6 h/d, 5 d/wk

Details on study schedule:

43 exposure days (64 study days) prior to initiation of mating, exposures were continued up to the day before sacrifice

Dose / conc.:

40 ppm (nominal)

Remarks:

0.143 mg/I; 40 +/- 2 ppm

Dose / conc.:

120 ppm (nominal)

Remarks:

0.428 mg/l; 116 +/- 8 ppm

Dose / conc.:

400 ppm (nominal)

Remarks:

1.428 mg/l; 386 +/- 19 ppm

No. of animals per sex per dose:

12 males/group

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: not specified
- Fasting period before blood sampling for clinical biochemistry: not specified

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- all animals: twice daily for mortality and gross clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Males: thorough physical examination once per week
- Females: not specified

BODY WEIGHT: Yes
- Males: once per week
- Females: on GD0, 15, 20

FOOD CONSUMPTION: not specified
WATER CONSUMPTION: not specified

OTHER:
After 25 exposure days (on day 39 of the study) blood was collected by orbital sinus bleeding from 5 randomly selected males in each treatment group and the control group. Serum was harvested and analyzed on a KDA (American Monitor Corp., Indianapolis, IN) for albumin, total protein, total bilirubin, alkaline phosphatase, and globulin (calculated). SGPT levels (serum glutamate-pyruvate-transaminase) were determined on a Centrifichem 400 (Union Carbide, Rye, NY). Standard manufacturer's methodology was used.

Oestrous cyclicity (parental animals):

not specified

Sperm parameters (parental animals):

not specified

Litter observations:

Mated females were sacrificed on GD20.

Postmortem examinations (parental animals):

- Males: Examination of external surface and tissues and organs of the abdominal, thoracic and scrotal cavities, measurement of liver and testes weight and their preservation in Bouin´s solution for microscopic evaluation
- Females: sacrifice on GD20, necropsy to examine tissues/organs as well as external surface of thoracic and abdominal cavities; determination of pregnancy status, counting corpora lutea and nidations; measurement of carcass weight (without gravid uterus)

Postmortem examinations (offspring):

Viablilty and weights of fetuses at GD20, examination for gross external abnormalities

Statistics:

- Body weights, organ weights, and clinical chemistry measurements: Dunnett's test (Dunnett, 1964)
- Nidation data: Mann-Whitney U-test (Siegel, 1956)
- Microscopic observations: Kolmogorov-Smirnov test or Fisher's exact test and the chi-square test (Siegel, 1956 ; Fisher, 1950 ; Berkson, 1978)
- Comparison of multiple treatments with control (except Dunnett's test): Bonferroni inequality (Miller, 1966).

Clinical signs:

no effects observed

Description (incidence and severity):

Male rats exhibited no significant treatment-related clinical signs of toxicity during exposure or nonexposure periods.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of males in treatment groups were not significantly different from control values.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical chemistry analyses (albumin, total protein, total bilirubin, globulin, and SGPT) indicated no significant differences betweenany of the treatment groups and the control group.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Microscopic evaluations were performed on testes and livers from all males and on kidneys that were noted to have apparent hydronephrosis at gross necropsy. There were no treatment-related changes observed microscopically in these organs. Random atrophy, which was observed in the testes of a few animals, was distributed with equal frequencies between the control and high-dose groups.

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

The reproductive performance data for males were copulations/number mated; copulations/exposure nights; copulations/mating pairs; number pregnant/number with confirmed copulation; mating efficiency and efficiency of males in effecting pregnancy. These data indicate no significant differences between any of the treatment groups and the control group in the number of confirmed copulations per mating opportunity or per female and no significant difference in the percentage of females with confirmed copulation that were pregnant.
Nidation and fetal data for females mated with DMAC exposed males showed no significant differences in pre- or postimplantation loss or fetal weights between females mated to treated males and females mated to control males. External examination of fetuses was generally unremarkable and no treatment-related effects were observed.

Dose descriptor:

NOAEC

Effect level:

400 ppm (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

Other effects:

no effects observed

Description (incidence and severity):

No significant differences were observed in pre- or postimplantation loss or fetal weights between females mated to treated males and females mated to control males. Gross examination of females and external examination of fetuses were generally unremarkable and no treatment-related effects were observed.

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

400 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatmend-related effects observed

Reproductive effects observed:

no

Lowest effective dose / conc.:

400 other: NOAEC = 400 ppm, no adverse effects on reproductive parameters examined up to the higest dose tested

Conclusions:

There were no effects on reproductive performance (mating efficiency) and on offspring after repeated subchronic inhalation exposure of male rats to dose levels up to 400 ppm (1400 mg/m³).

Executive summary:

In a male fertility study (Wang, 1989; Monsanto, 1982), male Sprague-Dawley rats were exposed via whole body inhalation (6h/d, 5d/wk for 15 weeks = 69 exposures) to N,N-dimethylacetamide (DMAC) and mated to untreated virgin females. Mean analytical exposure concentrations were 40 +/- 2, 116 +/- 8, and 386 +/- 19 ppm, respectively. A control group was exposed to air containing no DMAC. Clinical signs were recorded twice daily. Parameters analyzed in pups were viability, weight and external malformations. Necropsy was performed in exposed male animals as well as in untreated females. Testes and liver weights were measured and histopathology of liver, testes, and kidneys was performed. In female animals, mating efficiency was determined and nidations and corpora lutea studied. In addition, mating efficiency was recorded. No clinical signs and mortality were observed in parental animals. The only treatment-related effect was the change in absolute and relative liver weight of 12 and 22% noted at 120 and 400 ppm, respectively. Testes weights and testes/body weight ratios were not affected by treatment. Reproduction indices were not affected. The NOAEC for parental animals was set to 400 ppm, because liver weight gain was not accompanied by effects in clinical chemistry or treatment related histopathological changes. No treatment related effects were seen in pups. No clinical signs and mortality was observed. The NOAEC for pups (F1 generation) was concluded to be 400 ppm based on no observed adverse effects.

Reference 3

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Sperm abnormality test in male mice: Expoure to atmospheres containing 20 ppm or 700 ppm N,N-dimethylacetamide for 7 h per day for 5 consecutive days. Analysis of test atmospheres was by continuous infrared absorption monitoring at a wavelength of 9.9 μm. Mice were killed 5 weeks from the last day of dosing by neck dislocation. Sperm from the cauda epididymides was spread on slides, stained in 1% eosin and examined microscopically.

GLP compliance:

not specified

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
No details available.

Species:

mouse

Strain:

not specified

Sex:

male

Route of administration:

inhalation

Details on exposure:

GENERATION OF TEST ATMOSPHERE
The test atmospheres were produced by bubbling dry, oxygen-free nitrogen (BOC Limited) through a liquid reservoir of N,N-dimethylacetamide contained in a glass gas washing, or Drechsel bottle immersed in a temperature controlled water bath at 50°C. The nitrogen/N,N-dimethylacetamide vapour mixture so generated was ducted through 7/16", ID stainless steel piping to a glass mixing vessel and diluted with filtered, compressed air. The resultinq mixture of N,N-dimethylacetamide/air was ducted through 3/1" stainless steel piping to the top of the exposure chamber. The atmospheres in the exposure chambers were dynamic in that they were continuously generated for a single pass through the animal holding zone, before being extracted from the bottom and ducted away for 'scrubbing'. The required atmospheric concentrations within the exposure chambers were maintained by finely regulating the flow of nitrogen and diluting air into the mixing vessels, by means of adjustable flow meters.

HOMOGENEITY DATA
Before starting the animal expsures, chamber concentrations at both the high and low levels were determined by continuous monitoring for periods of up to 6 h. In addition, samples were measured from different areas (at least 9) of the animal holding zone to confirm uniformity of N,N-dimethylacetamide concentration. The maximum deviations encountered were +/-2.3% at the 20 ppm target concentration and +/- 2.4% at the 700 ppm target concentration.

MEASUREMENT OF CHAMBER CONCENTRATIONS
Continous monitoring during the 7 h exposure period from the breathing zone of the animals. Stainless steel sampling lines, fitted with a particulate filter (Whatman Mini-Filter, Grade 80) and positioned on a central reference point in each exposure chamber, were connected to the infra-red gas analysers. The sampling flow rate was approximately 4 l/min.
Calibration ranges adopted were 9.4-47.0 ppm (20 ppm target concentration) and 188-940 ppm (700 ppm target concentration).

TEST COMPOUND UTILISATION
The DMAC reservoir was replenished daily with test compound. Utilisation of test material was calculated on a daily basis by weighing the bottle before vapour generation began and deducting the weight of the bottle and remaining test compound on completion of the exposure period.

EXPOSURE PROCEDURE
Exposures were conducted during the 7 h of between approximately 09 .00 h and 16 .00 h on each exposure day. Animals were examined for any signs of ill health before and after exposure. They were not allowed access to food or water during the exposure period. Animal positions within the exposure chambers were rotated on a daily basis to minimise any possible exposure location variations. The chamber temperature and relative humidity were recorded at hourly intervals throughout the exposure period. The animals were also observed at regular intervals for the appearance of clinical signs or adverse reactions to treatment.

POSITIVE CONTROL
Dosing solutions were prepared daily 5 min before administration to the animals was started. The desired amount of ethyl methanesulphonate was weighed into a volumetric flask and diluted with distilled water to obtain the correct concentration. Positive control animals were not allowed access to food or water whilst the remaining test groups were being exposed. 200 mg/kg ethyl methanesulphonate was administered orally by gavage to the mice at a constant dose volume of 10 ml/kg at around 16.00 h for 5 consecutive days.

Details on mating procedure:

no mating

Duration of treatment / exposure:

Exposure period: 5 days
Premating exposure period (males): no mating
Premating exposure period (females): no mating
Duration of test: 6 weeks

Frequency of treatment:

7 h/d

Details on study schedule:

Expoure to atmospheres containing 20 ppm or 700 ppm N,N-dimethylacetamide for 7 h per day for 5 consecutive days. Mice were killed 5 weeks from the last day of dosing by neck dislocation. Sperm from the cauda epididymides was spread on slides, stained in 1% eosin and examined microscopically.

Dose / conc.:

0.07 other: mg/L

Remarks:

20 ppm

Dose / conc.:

2.53 other: mg/L

Remarks:

700 ppm

Control animals:

yes, concurrent no treatment

Positive control:

yes

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs of toxicity considered to be due to exposure to DMAC were observed in mice.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight values of the B6C3F1 mice taken at the time of dosing showed no effects upon mouse weight gain. EMS treated mice showed adversely affected body weights to some extent.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

SPERM ABNORMALITY TEST
There were no increases in the frequencies of abnormal sperm in any of the categories examined. The categories C and D, amorphous head and folded tail respectively, showed significantly higher frequencies in the EMS treated groups than in the air control group.

Reproductive performance:

not examined

Reproductive effects observed:

not specified

In a sperm abnormality test, groups of 10 male mice were exposed to the test substance for 5 consecutive days. No clinical signs of toxicity and no effects on body weight gain were observed. Sperm was  examined 5 weeks after the end of exposure. No significant differences in frequency of abnormal sperm between the exposed groups and controls were observed.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

1 080 mg/m³

Study duration:

subchronic

Species:

rat

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Quality of whole database:

The available dermal studies investigating reproductive effects on parental animals was conducted by Industrial Bio-Test Laboratories which was confirmed of engaging in extensive scientific misconduct, thus, the studies were disregarded for assessment.

Additional information

In a one-generation reproduction toxicity study (Ferenz et al., 1986; RL2: comparable to Guideline study but MTD also not reached; no histopathology of reproductive organs) 10 males and 20 females per group were exposed via the inhalation route 6 h per day, 5 days per week, for 10 weeks (prebreeding), then 7 days per week for 7-8 weeks (breeding, gestation, lactation) to 0, 30, 100, or 300 ppm (group I-IV, respectively; nominal concentrations corresponding to 0, 110, 360, 1080 mg/m³; analytical concentrations of 0, 31.1 ± 4.86, 101 ± 13.4, 291 ± 29.9 ppm). In additional groups (10 males and 20 females) only males (group V) or only females (group VI) were exposed to 300 ppm. The exposure period started when rats were 35 days old and ended for the males upon completion of the breeding period (after 63 exposures), and for the females and offspring at 21 days post partum (after 89-104 exposures). Half of the adult males in each group were sacrificed after the breeding period; the other half after a recovery period of 20 days. Dams were sacrificed on day 21 post partum. Mating started when rats were 100 days old (each male was placed in a cage with two females). Mating index, fertility index, males impregnating females (%), gestation length, and gestation index were not altered in any treatment group as well as viability index, lactation index and developmental parameters in F1 rats. In the P and F1 generation no treatment related clinical signs were observed and necropsy revealed also no treatment related effects. During the first 3 weeks of exposure a significant decrease in body weight was measured in P males of group V (300 ppm); this effect was not found in males of group IV (300 ppm) or in any other test group. No effects were found on absolute liver weights in P animals except a slight increase in treatment groups (not significant); in group IV the relative liver weight of males and females was significantly increased but not in group V and VI (also 300 ppm); no effects were reported in recovery males (one half of males of each group sacrificed after a post exposure observation period of 20 days). Body weight in exposed male and female F1 rats was significantly reduced at 300 ppm.

Conclusion: No effects on reproductive parameters in rats were found after repeated inhalation exposure to DMAC at a dose level of 300 ppm (1080 mg/m³).

In a male fertility study (Wang, 1989), male Sprague-Dawley rats were exposed via whole body inhalation (6h/d, 5d/wk for 15 weeks = 69 exposures) to N,N-dimethylacetamide (DMAC) and mated to untreated virgin females. Mean analytical exposure concentrations were 40 +/- 2, 116 +/- 8, and 386 +/- 19 ppm, respectively. A control group was exposed to air containing no DMAC. Clinical signs were recorded twice daily. Parameters analyzed in pups were viability, weight and external malformations. Necropsy was performed in exposed male animals as well as in untreated females. Testes and liver weights were measured and histopathology of liver, testes, and kidneys was performed. In female animals, mating efficiency was determined and nidations and corpora lutea studied. In addition, mating efficiency was recorded. No clinical signs and mortality were observed in parental animals. The only treatment-related effect was the change in absolute and relative liver weight of 12 and 22% noted at 120 and 400 ppm, respectively. Testes weights and testes/body weight ratios were not affected by treatment. Reproduction indices were not affected. The NOAEC for parental animals was set to 400 ppm, because liver weight gain was not accompanied by effects in clinical chemistry or treatment related histopathological changes. No treatment related effects were seen in pups. No clinical signs and mortality was observed. The NOAEC for pups (F1 generation) was concluded to be 400 ppm based on no observed adverse effects.

Both inhalation studies concerning effects for fertility were presented in the OECD SIDS document (2001; SIAM 13) on N,N-dimethylacetamide (DMAC), as well as in the CLH report "Proposal for Harmonised Classification and Labelling for N,N.Dimethylacetamide, CAS 127 -19 -5" submitted by the Netherland´s MSCA in November 2013.

Furthermore, the OECD SIDS document reported about a sperm abnormality test, where male mice were exposed to 20 and 700 ppm for 7 hours/day for 5 days/w for 6 weeks. They were not mated and sperm was analysed 5 weeks after the end of exposure. No differences in frequency of sperm abnormalities between exposed groups and controls were observed (NIOSH, 1980).

Three studies investigating reproductive performance in rats exposed via dermal and inhalation route are available (Monsanto 1973/1982). However, the studies were conducted by Industrial Bio-Test Laboratories which was confirmed of engaging in extensive scientific misconduct (OECD, 2005: Manual for the Assessment of Chemicals. Chapter 3: Data Evaluation). Therefore, the studies were not used for further assessment and disregarded due to the unreliability of the testing facility.

Waiver for the EOGRTS:

According to the REACH regulation (EC)1907/2006, Annex X, section 8.7.3, column 1 an EOGRTS with basic test design is a standard information requirement for substances manufactured or imported in quantities of 1000 tonnes or more. But "if a substance is known to cause developmental toxicity, meeting the criteria for classification as toxic for reproduction category 1A or 1B: May damage the unborn child (H360D), and the available data are adequate to support a robust risk assessment, then no further testing for developmental toxicity will be necessary. However, testing for effects on fertility must be considered." (REACH, Annex X, section 8.7, column 2)

DMAC, CAS 127-19-5 is listed in the Annex VI of Regulation (EC) No. 1272/2008 (CLP Regulation) with the classification for acute dermal and inhalation toxicity Cat. 4*; H312 and H332, and for developmental toxicity / teratogenicity Repr. 1B;H360D. As mentioned above, with this classification for developmental toxicity, an adaptation for further testing for effects on fertility might be possible.

1) Data base for classification or non-classification

Further testing for effects on fertility is considered to be scientifically not necessary for DMAC, as in a weight-of-evidence approach other information is available justifying the non-classification for effects on fertility:

1.1) Fertility studies

In particular, the one generation study according to OECD415 published by Ferenz and Kennedy (1986; RL2) did not reveal any adverse effects on fertility after inhalative exposure to rats up to high dose of 300 ppm (ca. 1080 mg/m3): No effects on reproductive performance (mating, fertility, gestation indices; viability and lactation indices), no changes in testes /ovaries weights in the parental generation. But at this dose systemic toxicity was observed, e.g. statistically significant decreased body weights in parental males and male and female pups.

In a male fertility study (Wang, 1989; RL2), male rats were exposed to 40, 120 and 400 ppm DMAC and mated to untreated females. A total of 69 exposure within 14 weeks produced treatment related effects of increased liver weights and liver/body weight ratios in the high- and medium-exposure groups of male rats without histopathological correlate. Testes weights were not affected and no effects were observed microscopically compared to controls. Reproductive data indicated no treatment related effects on copulation efficiency or efficiency in effecting pregnancy, and there were no detectable treatment-related effects on preimplantation loss, postimplantation loss, embrvotoxicity, or fetotoxicity.

Supportingly, in a sperm abnormality test, male mice were exposed to 20 and 700 ppm for 7 hours/day for 5 days/w for 6 weeks. They were not mated and sperm was analysed 5 weeks after the end of exposure. No differences in frequency of sperm abnormalities between exposed groups and controls were observed (NIOSH, 1980).

Kennedy reviewed in 2001, that DMAC did not repress fertility, when used as a cryoprotective agent for fowl spermatozoa.

1.2) Effects on reproductive organs in repeated dose toxicity studies

Overall, the available repeated dose toxicity studies do not indicate adverse effects on reproductive organs or tissues or reveal other concerns in relation with reproductive toxicity. In two oral repeated dose toxicity studies, reduced testes or uterus weights were observed in rats (Monsanto, 1979; BASF, 1976). However, this occurred at high doses (>= 1000 mg/kg), exceeding the MTD, determined by reduced body weights of at least -10%.

In two subacute inhalation studies, some testicular injury was mentioned in rats and mice (DuPont, 1983; Valentine, 1997a/b) at doses of 300 ppm, not exceeding the MTD. However, this was neither found in the above-mentioned fertility studies, nor in other RDT studies after subchronic or chronic exposure (Valentine, 1997c; Kinney, 1993; Malley, 1995). 

1.3) No need for an extension with cohort 1B, 2A/2B and/or 3

No extension with the cohort 1B (F2 generation) is considered necessary, as the substance has only industrial uses. The one professional use (ERC8a/PROC15) is located at industrial sites but included in the risk assessment because of the amounts used. Thus, significant exposure of consumers or professionals does not occur.

No extension with the cohorts 2A/2B (developmental toxicity) and/or 3 (developmental immunotoxicity) is considered necessary, as there are no particular concerns on (developmental) neuro- or immunotoxicity known; based on the available data.

In conclusion, the available information is considered sufficient justifying the non-classification for effects on fertility. The NOAEC for effects on fertility is 300 ppm (1080 mg/m3) in rats as no adverse effects were observed up to the highest dose tested in the one-generation study (Ferenz & Kennedy, 1986).

 

2) DNEL derivation and risk assessment

Developmental toxicity / teratogenicity was observed in rats, rabbits and mice after oral or inhalation exposure (see IUCLID chapter 7.8.2), resulting in the classification as reprotoxic Cat. 1B; H360D.

In several inhalative PNDT studies in rats maternal and developmental toxicity, e.g. decreased body weights, were observed at 300 ppm; malformations occurred at 450 ppm. Thus, the NOAEC for maternal and developmental toxicity in rats after inhalation exposure was 100 ppm (360 mg/m3). Similar results were obtained after oral administration at comparable doses (Dupont, 1997; Johannsen, 1987; BASF, 1976)

 

This NOAEC of 100 ppm (360 mg/m3) for maternal and developmental toxicity in rats is lower than the current NOAEC for fertility (300 ppm, the highest dose tested). Even if a further reproduction toxicity study would be performed, higher dosing is very unlikely, as mortality was observed at 622 ppm in rats in a subacute inhalation study (DuPont, 1983). Thus, detecting any adverse effects on fertility is in a further study is rather unlikely based on the available data.

With the available data a robust risk assessment is possible: The long-term DNELs for occupational exposure are based on the chronic NOAEC of 25 ppm (90 mg/m3) in rats and mice (Malley, 1995) and the subchronic dermal NOAEL of 94 mg/kg in dogs (Horn, 1961), ensuring an adequate risk assessment. 

Effects on developmental toxicity

Description of key information

RAT
ORAL: NOAEL = 65 mg/kg bw/d (maternal/offspring) (1997); OECD 414 and GLP
INHALATION: NOAEC: 100 ppm (360 mg/m³) (maternal toxicity and fetal toxicity) (Okuda, 2006; Salomon, 1991); Equivalent or similar to 414, GLP (unknown)
DERMAL: no study available due to major methodological deficiencies.

MOUSE
ORAL: NOAEL= 400 mg/kg bw/d (maternal toxicity and developmental toxicity), 240 mg/kg bw/d (teratogenicity) (BASF, 1976); Equivalent or similar to 414 (1973), non-GLP

RABBIT
ORAL: NOAEL = 94 mg/kg bw/day (maternal developmental toxicity and teratogenicity); According to 414 (1976); Equivalent or similar to 414 (1974), both non-GLP
INHALATION: NOAEC (maternal) = 2.0 mg/l, NOAEC (developmental toxicity) = 0.2 mg/l (Klimish & Hellwig, 2000 = BG Chemie, 1989); According to 414, GLP
DERMAL: no study available due to major methodological deficiencies.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

13-14 pregnant rabbits per group; exposure restricted to gestation days (GD) 7-19

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

13-14 pregnant rabbits per group; exposure restricted to gestation days (GD) 7-19

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
- Source: BASF AG
- Purity: 99.9%
- Batch No.: 86/104
No further details available.

Species:

rabbit

Strain:

other: Russian

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach/Riss, Germany
- Mean body weight at initiation: 2.3 kg
- Age at initiation: 23-27 weeks old
- Acclimation period: Prior to artificial insemination
- Housing: Individually
- Certified diet and tap water: ad libitum (water and diet analysed for contaminations, negative results)

ENVIRONMENTAL CONDITIONS:
- Temperature: 20-24 °C
- Humidity: 30-70 %
- Photoperiod: 12 hours dark/12 hours light

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION /EXPOSURE PARAMETERS
The animals were adapted to exposure conditions (air without test substance) at gestation days 1-4.
Vapourisation was performed at a temperature of 50, 60, or 65°C (low, mid, and high dose, respectively) in an evaporator resulting in a mixture with air (50 % relative air humidity, 22 °C; compressed air in high dose group); the vapour-air-mixture was discharged into the exposure chamber (ca. 1.1 m³). There was negative pressure for treatment groups and positive pressure for the control group. Technical parameters of exposure (temperature, pressure, air humidity, air flow) were controlled.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Measurement of total hydrocarbon by GC technique (calibrated); daily intermittend sampling.
Temperature (23.5, 24.7, 24.6, 25.3 °C at 0, 0.2, 0.7, 2 mg/L) and relative air humidity (53 %) were kept constant.

Details on mating procedure:

- Impregnation procedure: Artificial insemination

Duration of treatment / exposure:

Gestation days (GD) 7-19

Frequency of treatment:

Daily for 6 h/day

Duration of test:

GD 29

Dose / conc.:

0.2 mg/L air (analytical)

Remarks:

± 0.01 mg/l (SD). 0.2 mg/l (nominal concentration).

Dose / conc.:

0.7 mg/L air (analytical)

Remarks:

± 0.01 mg/l (SD). 0.7 mg/l (nominal concentration).

Dose / conc.:

2 mg/L air (analytical)

Remarks:

± 0.06 mg/l (SD). 2.0 mg/l (nominal concentration).

No. of animals per sex per dose:

Initial 15 rabbits per group.
In satellite groups (control and high dose) 5 pregnant rabbits.

Control animals:

yes, concurrent no treatment

Details on study design:

Dose selection rationale: No details

Maternal examinations:

CLINICAL SIGNS, MORTALITY:
Clinical signs were recorded daily during, prior and after exposure (mortality).

BODY WEIGHT:
Body weight was measured on GD 3, 7, 10, 13, 16, 19, 21, 24, 27 and 29.
Corrected body weight gain was measured (substracting uterus weight).

GROSS PATHOLOGY:
At termination a necropsy was performed.

OTHER:
In satellite groups (control and high dose; n=5) additional clinical chemistry parameters were assessed (blood sampling at GD 20) and histopathology of the liver was performed; clinical chemistry parameters included alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, inorganic phosphate, calcium, glucose, total protein, bilirubin, triglyceride.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination.
Examinations included:
- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of resorptions (temporally differenciated), dead and living fetuses

Fetal examinations:

Fetal weight, sex, viability and placental weight were recorded (fetal length was not measured .

Retardations and variations were recorded as well as malformations, based on:
- External examinations
- Soft tissue examinations
- Skeletal examinations (via x-rays)
- Head examinations (12 transversal sections after fixation in Bouin's solution)

Statistics:

WILLIAMS test
FISHER exact test
KRAUTH test
NOVA-DUNNETTS test
Level of significance: p<0.05
Clinical chemistry: KRUSKAL-WALLIS, MANN-WHITNEY U-Tests

Indices:

See results

Historical control data:

Yes

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

There was one abort in the low dose group on GD 23 (dam sacrificed).

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Pre- and post-implantation loss:

no effects observed

Dead fetuses:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

There were effects on mean uterus weight (increased at 0.2 mg/l and 0.7 mg/l level) related to the low number of fetuses in the control group (compared to historical controls). Furthermore, placental weight was reduced in treatment groups, but this effects was also related to the reduced number of living fetuses in the control group (see also placental weight/litter).

In each group one rabbit was not pregnant (conception rate 93.3 % in all groups).

Dose descriptor:

NOAEC

Effect level:

2 mg/L air

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

mortality

Dose descriptor:

NOAEC

Effect level:

2 mg/L air

Based on:

test mat.

Basis for effect level:

dead fetuses

pre and post implantation loss

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

2.0 mg/l: Reduced fetal and placental weights were observed.
0.7 mg/l: Effects on fetal weight in these dose groups were considered not to be of toxicological relevance (low number of fetuses in control group and increased litter weight in treatment groups)

Changes in sex ratio:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

2.0 mg/l: Increased variations (skeletal variations of ribs, sternum and vertebral column; separated origin of the carotids; also increased compared to historical controls) were observed.

0.7 mg/l: Increased skeletal variations, e.g. sternebrae fused and accessory ribs, both outside historical range, but no statistically significant increase in total skeletal variations. Increases in skeletal retardations (not dose related) were within the historical range.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Increased malformations (not significant but uncommon in historical controls like malformations of the vasculature; increased incidence in defects of the septum) were considered by the authors to be treatment related.

Dose descriptor:

NOAEC

Effect level:

0.2 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: increased skeletal variations

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: rib

other: skeletal: sternum, vertebral column; visceral: vasculature, defects of the septum

Developmental effects observed:

yes

Lowest effective dose / conc.:

700 mg/m³ air (analytical)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

no

Developmental toxicity in rabbits after inhalation exposure to N,N-dimethylacetamide

Data related to main groups

Parameter	Vehicle control	0.2 mg/L	0.7 mg/L	2 mg/L
Pregnant rabbits	14/15	14/15	14/15	14/15
Rabbits with abort	-	1	-	-
Implantations/dam	6.0	6.92	7.50	6.71
Living fetuses/dam	4.71	6.54	6.71	5.57
Dead implants/dam	1.29	0.38	0.79	1.14
% post-implantation loss	23.1	6.0	13.3	17.9
Placental weight m&f (g)	4.94	4.23**	4.22**	4.03**
Placental weight/litter (g)	24.4	27.2	27.3	21.8
Litter weight (g)	206.7	244.3	252.5	197.9
Fetal weight m and f (g)	41.3	37.5*	38.4*	35.9**
% fetuses/litter with soft tissue malformations	6.4	2.1	3.2	13.6
% fetuses/litter with soft tissue variations	48.4	30.4	40.9	85.0*
% fetuses/litter with skeletal variations	9.2	8.0	23.8	61.6**

*: p<0.05; **: p<0.01

Conclusions:

The NOAEC for developmental effects in rabbits after inhalation exposure was 0.7 mg/L and the LOAEC 2 mg/L; no maternal toxicity was found at dose levels up to 2 mg/L.

Executive summary:

The test substance was investigated for its teratogenic potential in female Humalayan rabbits exposed by inhalation (study report of BG Chemie, 1989; published by Klimish and Hellwig, 2000). Fifteen animals per dose group and control group were used and exposed to 0.2, 0.7, and 2.0 mg/l (equivalent to 0, 57, 200 or 570 ppm) for 6 hours/day from GD7-19 and sacrified at GD29. Two satellite groups exposed to 0 or 2.0 mg/L with 5 animals per group were observed for clinical signs, lethality and body weight until GD20 and clinical chemistry, gross pathology and histopathological investigation of the liver were performed. Maternal animals of the main groups were observed for clinical signs, body weight, lethality, weight of uterus, gross pathology, implants, and corpora lutea. Fetuses were examined for number, weight, sex, weight of placenta, external observations, soft tissue observations including observations of the head, and skeletal observations.

Maternal toxicity was not obseved neither in main groups nor in satellite groups.

Fetotoxic effects were caused at a concentration of 0.7 mg/L (increased skeletal variations, e.g. fused sternebrae and accessory ribs), but there was no statistically significant increase in total skeletal variations. At 2.0 mg/L fetotoxic effects (e.g., significantly decreased fetal and placental weights, increase in soft tissue and skeletal variations) and also signs of a weak teratogenic effect expressed as a marginal, statistically not significant increase in soft tissue malformations (regarding the heart and great vessels) were observed. No compound-related effects were observed in the fetuses after exposure to 0.2 mg/l.
Thus, the highest concentration tested under these conditions (2.0 mg/1) was found to be a no-observable-adverse-effectlevel (NOAEL) for the maternal Himalayan rabbit, whereas 0.2 mg/L was defined as the NOAEL for the developing organism.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

treatment period gestation day 6-15; partly limited documentation, e.g. no details about the test substance; untreated controls; no historical control data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

treatment period gestation day 6-15

Principles of method if other than guideline:

The test was performed according to FDA Guidelines for reproduction studies for safety evaluation of drugs for human use. Food and Drug Administration, Washington, 1966.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
- Substance type: technical product
- Substance No.: XX/12
No further details available.

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wiga, Sulzfeld, Germany
- Housing: 2 animals per cage
- Certified diet and tap water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 50-60 %
- Photoperiod: 12 hours dark/12 hours light

Only females with vaginal plug were used (gestation day 0 [GD 0]).

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared daily.

Constant application volume in all dose groups: 5 mL/kg bw
Dose was related to the initial weight at GD 0.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: overnight
- Proof of pregnancy: [vaginal plug] referred to as day 0 of pregnancy (GD 0).

Duration of treatment / exposure:

Gestation days (GD) 6-15

Frequency of treatment:

Once daily

Duration of test:

Termination at GD 20, caesarean section

Dose / conc.:

106 mg/kg bw/day (actual dose received)

Remarks:

corresponding to 113 µl/kg bw/day

Dose / conc.:

320 mg/kg bw/day (actual dose received)

Remarks:

corresponding to 340 µl/kg bw/day

Dose / conc.:

960 mg/kg bw/day (actual dose received)

Remarks:

corresponding to 1020 µl/kg bw/day

No. of animals per sex per dose:

18-24 pregnant animals

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Data from acute oral toxicity in female rats (LD50: 5100 µL/kg bw).
- Rationale for animal assignment: Randomisation

Maternal examinations:

CLINICAL SIGNS:
Clinical symptoms were recorded daily.

BODY WEIGHT:
Body weight was measured thrice weekly.

GROSS PATHOLOGY:
A necropsy was conducted at termination.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Placenta weight of living fetuses: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Dead and living fetuses: Yes

Fetal examinations:

Weight, length, and sex of fetuses, and placental weight were determined.

- External examinations: all per litter
- Soft tissue examinations: 1/3 of all fetuses was processed for the assessment of visceral effects (fixation in Bouin's solution for 2 weeks, 15 transversal sections).
- Skeletal examinations: 2/3 of all fetuses was processed for the assessment of skeletal effects (Dawsen-method; Alizarin staining).

Statistics:

Analysis of variance
t-test
Level of significance: p<=0.05

Indices:

See results.

Historical control data:

No data available.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 340 and 1020 µl/kg, five and six animals showed vaginal bleeding, respectively. Other clinical signs demonstrating toxicity were not observed. No effects reported in the 113 µl/kg goup.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Animals receiving 340 and 1020 µl/kg showed a reduction in body weight gain. A dose-response relationship was observed. Body weight gain of animals receiving 113 µl/kg was comparable to control group.

Gross pathological findings:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

Implantations were similar in all groups. At 1020 µl/kg, all fetuses died mostly in post-implantation phase. Significantly increased dead implants were seen in the 340 µl/kg group. Embryos died mostly in the pre-implantation phase. A dose-response relationship was noted. No adverse effects were observed in the 113 µl/kg group.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

All fetuses died in the 1020 µl/kg group.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Weight of placenta was significantly reduced in the 340 µl/kg group. No difference to control was observed in the 113 µl/kg group.

Dose descriptor:

NOAEL

Effect level:

106 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

Dose descriptor:

NOAEL

Effect level:

106 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

Implantation loss

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

No living fetuses were observed in the highest treatment group (1020 µl/kg). At 340 µl/kg, body weight was significantly reduced. At 113 µl/kg, body weight was significantly enhanced.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

At 1020 µl/kg, no living fetuses were observed (0%). At 340 µl/kg, the average number of living fetuses was slightly reduced (88.59%).

External malformations:

effects observed, treatment-related

Description (incidence and severity):

At 340 µl/kg, six fetuses showed anasarca, two showed aplasia of the tail and one fetus showed atresia ani. At 113 µl/kg, only one fetus showed malformations (aplasia of the tail and atresia ani). A dose-response relationship was observed.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

At 340 µl/kg, eighteen fetuses showed significant malformations observed in the spine (split and aplasia of vertebrae). Variations and retardations were seen to an increased amount. At 113 µl/kg, variations and retardations were similar to control.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

At 340 µl/kg, two fetuses showed malformations in ureter (hydroureter).

Details on embryotoxic / teratogenic effects:

Eighteen fetuses showed significant malformations (=6.82%) in the 340 µl/kg group.

Dose descriptor:

NOAEL

Effect level:

106 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

external malformations

skeletal malformations

Abnormalities:

effects observed, treatment-related

Localisation:

external: tail

external: anus

skeletal: vertebra

visceral/soft tissue: urinary

other: external: skin

Developmental effects observed:

yes

Lowest effective dose / conc.:

320 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Developmental toxicity in rat after oral application of N,N-dimethylacetamide

Parameter	Control 1 (untreated)	106 mg/kg bw/day	Control 2 (untreated)	320 mg/kg bw/day	Control 3 (untreated)	960 mg/kg bw/day
Number of litters	18	18	22	24	23	20
Implants per litter	12.8	12.4	13.4	12.4	12.6	11.1
% dead implants	3.0	6.2	5.7	11.4*	8.9	100
% living fetuses related to implantations	97.0	93.7	94.3	88.6	91.1	Complete resorption
No. of male/female fetuses	111/112	103/97	135/144	138/126	141/124	0/0
Living fetuses per litter	12.4	11.7	12.7	11.0	11.5	-
Fetal weight in g	3.71+-0.29	4.10+-0.29^1	3.84+-0.29	3.14+-0.38*	3.27+-0.25	-
Fetal length in cm	3.60+-0.12	3.72+-0.12^1	3.84+-0.13	3.47+-0.23	3.65+-0.15	-
Placental weight in g	0.52+-0.08	0.52+-0.08	0.57+-0.08	0.47+-0.07*	0.53+-0.07	-
% fetuses with malformations	0	0.5	0.7	6.8*	4.9	-
No. of runts	1	1	1	50	0	-

f: females; m: males; *: significant, p<0.05

1: significantly increased compared to the concurrent untreated control (no toxicological relevance)

Means ± standard deviation given (not available for all parameters)

Conclusions:

Developmental toxicity was found in rats at oral dose levels without maternal toxicity; the LOAEL was 320 mg/kg bw/day and the NOAEL for developmental toxicity was 106 mg/kg bw/day. For maternal toxicity the NOAEL was 960 mg/kg bw/day.

Executive summary:

The test substance was investigated for its teratogenic potential using rats after ten-times oral application. Investigations were conducted according to guidelines set forth by FDA (Guidelines for reproduction studies for safety evaluation of drugs for human use, Food and Drug Administration, Washington, 1966).

The LD50 calculated, applying the methods proposed by Litchfield and Wilcoxon, was used for the generation of the test concentrations. Once applied orally and after an observation period of 14 days, the LD50 in the rat was 5100 (4470 -5820) µl/kg bw. Test concentrations chosen for the current study were 1020, 340, and 113 µl/kg bw corresponding to 1/5, 1/15, and 1/45 LD50, respectively. Untreated control groups were included.

The oral application of the test substance diluted in destilled water was performed using a cannula from Day 6 through Day 15 post coitum (total applications: ten). The application volume was 500 µl/100 g animal/day.

Caesarean section was performed on Day 20 post coitum. All fetuses were observed for external malformations. Skeletal and visceral malformations as well as variations and retardations were examined on 2/3 and 1/3 fetuses of each litter, respectively.

Maternal animals receiving 1020 µl/kg bw had a reduced body weight gain. No clinical signs of toxicity were observed. 1020 µl/kg bw were fetal toxic. Embryos died mostly in the medium-late gestation period. No living fetuses were observed.

At 340 µl/kg bw, reduced body weight gain was noted after Day 11. The test concentrations was weak fetal toxic. Fetuses died in the early and medium-late gestation period. Reduced fetal body weight and an increase in skeletal variations and retardations were concluded to be fetal toxic. A weak teratogenic effect was observed. The amount in percent of fetuses with malformations was statistically significant. Malformations were considered to be treatment-related based on the type of malformations observed.

At 113 µl/kg bw, no significant increase in dead fetuses was observed. No clinical signs were reported. The test concentration did not induce fetal toxicity. Body weight as well as skeletal and visceral retardations and variations were similar to the control group. No teratogenic effect was observed.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1970 - 1973

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

no details about the test substance; data restricted to effects after single applications

Qualifier:

no guideline followed

Principles of method if other than guideline:

Prenatal toxicity of dimethylacetamide in the mouse after single oral application.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL
- Source: BASF AG, Germany
- Appearance: colorless liquid
- purity: technical grade

Species:

mouse

Strain:

NMRI

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Firma WIGA, Sulzfeld, Germany
- Housing: five animals per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Constant application volume: 200 µL per mouse

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: Females were paired with fertile males of the same stock
- Length of cohabitation: 2 h (8 am to 10 am)
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug. This day was termed as Day 0.

Duration of treatment / exposure:

Single application via gavage at gestation days (GD) 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15

Frequency of treatment:

Single application

Duration of test:

Gestation day (GD) 18

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Remarks:

427 µl/kg (actual ingested)

Dose / conc.:

600 mg/kg bw/day (actual dose received)

Remarks:

640 µl/kg (actual ingested)

Dose / conc.:

1 200 mg/kg bw/day (actual dose received)

Remarks:

1280 µl/kg (actual ingested)

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

Remarks:

3200 µl/kg (actual ingested)

No. of animals per sex per dose:

>20 mice

Control animals:

yes, concurrent no treatment

Details on study design:

0, 400, 600, 1200, or 3000 mg/kg bw at GD 9
0, 600, 1200, or 3000 mg/kg bw at GD 8, 10, 11, 12
1200 or 3000 mg/kg bw at GD 6, 7, 13, 14
3000 mg/kg bw at GD 15

Groups of pregnant mice were treated once on a single day during gestation to various doses of the test substance. It is suggested that the aim was to establish the most sensitive time point for fetal toxicity, embryo lethality and or teratogenicity by exposing pregnant females on either day between day 6 and day 15 of the gestation period.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

MORTALITY: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: three times a weak

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 18
- Organs examined: uterus
- Gros pathology

OTHER:
- Implantation sites and resorptions
- Living and dead fetuses
- From living fetuses, sex, body weight, weight of placenta, and length was determined

Ovaries and uterine content:

The ovaries and uterine content was examined after termination.
Examinations included:
- Number of Implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Placental weight: Yes

Fetal examinations:

Fetal weight, length and sex were determined as well as the number of living and dead fetuses.
Teratogenic effects were recorded.

- External examinations: all per litter
- Soft tissue examinations: 1/3 of all fetuses was processed for the assessment of visceral effects (fixation in Bouin's solution for 2 weeks, 15 transversal sections)
- Skeletal examinations: 2/3 of all fetuses was processed for the assessment of skeletal effects

Statistics:

Analysis of variance (body weight and length of fetuses, weight of placenta); when significant (S ≥ 95%) a t-test was performed, significant level was + ≥ 95%, ++ ≥ 99%. Chi square test (S ≥ 95%) was used for dead implants and malformations. When chi sqaure test was significant, a fourfold test was performed (* ≥ 95%; ** ≥ 99%).

Indices:

No data

Historical control data:

No data

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Day 8: At 640 µl/kg, one animal had an abortion.
Day 14: One animal in the 3,200 µl/kg group had an abortion.
Day 15: Two animals treated with 3,200 µl/kg had an abortion.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed throughout the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Day 9: At 3,200 µl/kg, body weight gain was reduced.
Day 10: At 3,200 µl/kg, body weight gain was reduced.
Day 14: Reduced body weight gain in 3,200 µl/kg group.

Food consumption and compound intake (if feeding study):

not examined

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

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

Day 8: At 640 µl/kg, one animal had an abortion.
Day 14: One animal in the 3,200 µl/kg group had an abortion.
Day 15: Two animals treated with 3,200 µl/kg had an abortion.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

Day 6: At 3,200 µl/kg, dead implants were significantly increased (99%).
Day 8: At 3,200 µl/kg, dead implants were significantly increased (99%).
Day 9: At 3,200 µl/kg, dead implants were significantly increased (99%) and at 640 µl/kg significantly reduced (99%).
Day 10: At 3,200 µl/kg, dead implants were significantly increased (99%). At 1,280 µl/kg, increase of dead implants was not statistically significant.
Day 13: At 3,200 µl/kg, dead implants were significantly increased (99%).
Day 14: At 3,200 µl/kg, dead implants were increased although not significantly.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Day 12: The number of implantations was reduced at 3,200 µl/kg.

Dose descriptor:

NOAEL

Effect level:

1 200 other: mg/kg bw

Based on:

test mat.

Basis for effect level:

dead fetuses

Dose descriptor:

NOAEL

Effect level:

1 200 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

The number of implantations was reduced on gestation day 12 at 3,200 µl/kg bw and the number of dead fetuses was increased on all treated gestation days at 3,200 µl/kg bw.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Day 6: At 1,280 or 3,200 µl/kg, the body weight was reduced significantly. Number of runts not increased.
Day 8: At 3,200 µl/kg, the body weight was reduced significantly.
Day 9: At 3,200, 1,280, or 640 µl/kg, the body weight was reduced significantly (99%). A dose-response relationship was noted. At 427 µl/kg the body weight was significantly increased (99%).
Day 10: All doses (640, 1,280, and 3,200 µl/kg) led to a significant reduction in body weight (99%).
Day 11: All doses (640, 1,280, and 3,200 µl/kg led to a significant reduction in body weight (99%). Number of runts was increased in all animals.
Day 12: All doses (640, 1,280, and 3,200 µl/kg) led to a significant reduction in body weight (99% at 1,280 and 640 µl/kg and 95% at 3,200 µl/kg).
Day 13: At 3,200 µl/kg, the body weight was reduced significantly.
Day 14: At 3,200 µl/kg, the body weight was reduced significantly (99%).
Day 15: At 3,200 µl/kg, the body weight was reduced significantly (99%).

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

Day 6: At 3,200 µl/kg, average number of fetuses was reduced (73.59%; number of fetuses based on implantations).
Day 8: At 3,200 µl/kg, average number of fetuses was reduced (66.24%; number of fetuses based on implantations).
Day 9: At 3,200 µl/kg, average number of fetuses was reduced (52.75%; number of fetuses based on implantations).
Day 10: At 3,200 or 1,280 µl/kg, average number of fetuses was reduced (64.04% or 77.83%, respectively; number of fetuses based on implantations).
Day 13: At 3,200 µl/kg, a reduced number of living fetuses were reported (77.86%; number of fetuses based on implantations).
Day 14: At 3,200 µl/kg, a reduced number of living fetuses were reported (73.93%; number of fetuses based on implantations).
Day 15: At 3,200 µl/kg, a reduced number of living fetuses was reported (77.04%; number of fetuses based on implantations).

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Day 6: At 3,200 µl/kg, malformations to the head (total exencephaly, micrognathia, anophthalmia, and total skull malformation). One fetus in the control had a bent tail. At 1,280 µl/kg and corresponding control, one fetus each had total exencephaly.

Day 7: At 3,200 µl/kg, six fetuses had total exencephaly, three fetuses had brachygnathia inferior, two had total skull malformation, three had macroglossia and two fetuses showed each exophthalmia and or anophthalmia. No malformations were observed in the corresponding control. At 1,280 µl/kg, three fetuses had a bent tail and three fetuses had exencephaly. Only one fetus in the corresponding control group ha total exencephaly.

Day 8: At 3,200 µl/kg, total exencephaly, micrognathia, spina bifida, kyphosis and oligodactyly, cleft palate, tail shortenings, and split of the upper jaw. No fetuses of the control group had malformations. At 1,280 µl/kg, twenty-nine fetuses had total exencephaly, one fetus each had partial exencephaly, exophthalmia, and cleft palate. One fetus had a bent tail. Only one fetus of the control group had total exencephaly. At 640 µl/kg, only one fetus had total exencephaly. No malformations in the corresponding control group was observed.

Day 9: At 3,200 µl/kg, exencephaly, syndactyly, oligodactyly, polydactyly, and accessory toes were observed. Two fetuses of the control group had bent tail. At 1,280 µl/kg, total exencephaly and scoliosis was observed twelve-times and one-time, respectively. One fetus of the corresponding control group had a bent tail. At 640 µl/kg, four fetuses had total exencephaly. No malformations in the corresponding control group was observed. At 427 µl/kg, three fetuses had total exencephaly. One fetus of the corresponding control group had brachygnathia inferior, hernia umbilicalis and split of the upper jaw.

Day 10: At 3,200 µl/kg, oligodactyly and syndactyly were observed. No fetuses of the control group had malformations. At 1,280 µl/kg, no malformations were reported. Two fetuses had a bent tail of the control group. At 640 µl/kg, only one fetus had total exencephaly. One fetus had total exencephaly and one fetus had bent tail in the corresponding control group.

Day 11: At 3,200 µl/kg, oligodactyly, syndactyly, and accessory toes were observed. One fetus of the control group had bent tail. At 1,280 µl/kg, one fetus had total exencephaly. No malformations were reported in the control group. At 640 µl/kg, two fetuses had bent tail. Two fetuses had total exencephaly in the corresponding control group.

Day 12: At 3,200 µl/kg, oligodactyly, polydactyly, and aplasia of the claw were observed. Three fetuses of the control group had bent tail. At 1,280 µl/kg, five fetuses had oligodactyly and one fetus had bent tail. No malformations were reported in the corresponding control group. At 640 µl/kg, one fetus each had total exencephaly, ectopia visceralis, and bent tail. One fetus had total exencephaly and one had bent tail in the corresponding control group.

Day 13: At 3,200 µl/kg and the corresponding control group, no malformations were observed. At 1,280 µl/kg, two fetuses had total exencephaly. No malformations were reported for the corresponding control group.

Day 14: At 3,200 µl/kg, one fetus had total exencephaly and a shortening of toes. One fetus of the corresponding control group had exencephaly. At 1,280 µl/kg, one fetus had bent tail. One fetus had total exencephaly in the corresponding control group.

Day 15: At 3,200 µl/kg, one fetus had partial exencephaly. No malformations were observed in the corresponding control group.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Day 6: At 3,200 µl/kg, eighteen fetuses (8.61%) had malformations to the skull (exencephaly, micrognathia, internal hydrocephalus, cleft palate). Variations and retardations were not treatment-related. No fetuses of the control group had malformations. At 1,280, six fetuses (2.47%) had malformations to the skull (cleft palate) although findings were not significant. Variations and retardations were not treatment-related. Four fetuses (1.85%) of the corresponding control group had malformations affecting the skull and the ribs (fused ribs).

Day 7: At 3,200 µl/kg, twenty-two fetuses (13.10%) had malformations to the skull, limbs, ribs, and spine which were significantly increased. Variations and retardations were treatment-related. Four (3.17%) malformations concerning the skull were observed in the corresponding control group. At 1,280, five fetuses (2.69%) had malformations to the skull and ribs. Variations and retardations were not treatment-related. Four fetuses (1.85%) of the corresponding control group had malformations affecting the skull and the ribs.

Day 8: At 3,200 µl/kg, 131 fetuses (84.52%) had significantly increased malformations to the skull, spine, ribs, and kidneys. Variations and retardations were treatment-related. Two (0.82%) fetuses of the control group had malformations to the skull. At 1,280, 36 fetuses (17.14%) had significantly increased (99%) malformations to the skull, spine, and ribs. Variations and retardations were treatment-related. Four fetuses (2.55%) had malformations to skull and ribs in the 640 µl/kg group which was not significant. Variations and retardations were not treatment-related. Two fetuses (1.41%) of the control group had malformations to the skull and ribs.

Day 9: At 3,200 µl/kg, 75 fetuses (65.22%) had significantly increased malformations to the skull, spine, ribs, and limbs. Variations and retardations were treatment-related. Only 0.81% fetuses of the control group had malformations to the skull. At 1,280, 115 fetuses (61.50%) had significantly increased (99%) malformations to the skull, spine, and ribs. Variations and retardations were increased. Five fetuses (2.50%) had malformations to skull and ribs in the corresponding control group. At 640 µl/kg, 22 fetuses had significantly increased malformations to the skull and ribs. Variations and retardations were not treatment-related. Two fetuses (1.41%) of the control group had malformations to the skull and ribs. At 427 µl/kg, 4 fetuses (1.33%) had malformations to the skull and ribs which were not significant. Variations and retardations were not treatment-related. Six fetuses (2.42%) of the corresponding control group had malformations to the skull and ribs.

Day 10: At 3,200 µl/kg, 45 fetuses (34.62%) had significantly increased malformations to the skull and limbs. Variations and retardations were not increased. One (0.65%) fetus of the control group had a malformation to the skull. At 1,280, 13 fetuses (7.88%) had significantly increased (95%) malformations to the skull and spine. Variations andretardations were not treatment-related. Five fetuses (2.50%) had malformations to skull and ribs in the corresponding control group. At 640 µl/kg, six fetuses (2.38%) had malformations to the skull and ribs which was not statistically significant. Variations and retardations were not treatment-related. Five fetuses (1.78%) of the control group had malformations to the skull, ribs, and spine.

Day 11: At 3,200 µl/kg, 132 fetuses (61.97%) had significantly increased malformations to the skull and limbs. Variations and retardations were not increased. One (0.47%) fetus of the control group had a malformation to the skull. At 1,280, five fetuses (2.82%) had malformations to the skull which were not significant. Variations and retardations were not treatment-related. Five fetuses (2.22%) had malformations to skull in the corresponding control group. At 640 µl/kg, eight fetuses (3.31%) had malformations to the skull and ribs which were not statistically significant. Variations and retardations were not treatment-related. Five fetuses (2.22%) of the control group had malformations to the skull.

Day 12: At 3,200 µl/kg, 31 fetuses (24.03%) had significantly increased malformations to the skull and limbs. Variations and retardations were not treatment-related. Three (1.78%) fetuses of the control group had malformations to the spine. At 1,280, fifteen fetuses (7.32%) had malformations to the skull, ribs, and limbs which were not significant. Variations and retardations were not treatment-related. Six fetuses (3.43%) had malformations to skull and ribs in the corresponding control group. At 640 µl/kg, four fetuses (1.94%) had malformations to the skull and ribs which were not statistically significant. Variations and retardations were not treatment-related. Five fetuses (1.78%) of the control group had malformations to the skull and ribs.

Day 13: At 3,200 µl/kg, one fetus (0.47%) had a malformation to the skull which was not significant. Variations and retardations were not treatment-related. No fetuses of the control group had malformations. At 1,280, five fetuses (2.69%) had malformations to the skull and ribs although findings were not significant. Variations and retardations were not treatment-related. Six fetuses (3.43%) of the corresponding control group had malformations affecting the skull and ribs.

Day 14: At 3,200 µl/kg, five fetuses (3.21%) had a malformation to the skull which was not significant. Variations and retardations were not treatment-related. Five fetuses (4.07%) of the corresponding control group had malformations to the skull. At 1,280, three fetuses (1.46%) had malformations to the skull, spine, and ribs although findings were not significant. Variations and retardations were not treatment-related. Five fetuses (2.46%) of the corresponding control group had malformations affecting the skull.

Day 15: At 3,200 µl/kg, one fetus (0.66%) had a malformation to the skull which was not significant. Variations and retardations were not treatment-related. Two fetuses (1.11%) of the corresponding control group had malformations to the skull.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

LENGTH OF FETUSES:

Day 6: At 1,280 or 3,200 µl/kg, length of fetuses was significantly reduced.
Day 7: At 1,280 only, length of fetuses was significantly reduced.
Day 8: At 640, 1,280, or 3,200 µl/kg, length of fetuses was significantly reduced.
Day 9: At 640, 1,280, or 3,200 µl/kg, length of fetuses was significantly reduced.
Day 10: All doses (640, 1,280, and 3,200 µl/kg) led to a significant reduction of length (99%).
Day 11: 3,200 or 1,280 µl/kg led to a significant reduction of length (99%).
Day 12: 1,280 or 640 µl/kg led to a significant reduction of length (99%).
Day 13: At 1,280 or 3,200 µl/kg, length of fetuses was significantly reduced.
Day 14: At 3,200 µl/kg, length of fetuses was significantly reduced (99%) and at 1,280 µl/kg length was increased significantly (99%).
Day 15: At 3,200 µl/kg, length of fetuses was significantly reduced (99%).

WEIGHT OF PLACENTA:

Day 6: Was increased significantly both at 1,280 and 3,200 µl/kg.
Day 7: Was increased significantly at 3,200 µl/kg.
Day 8: Was reduced significantly at 3,200 µl/kg (99%). At 1,280 µl/kg, the weight was increased significantly (95%).
Day 9: Was reduced significantly at 3,200 µl/kg (99%).
Day 10: Was reduced significantly at 3,200 µl/kg or 1,280 µl/kg.
Day 11: Was reduced significantly at 3,200 µl/kg or 1,280 µl/kg (99%).
Day 12: Was reduced significantly at 1,280 µl/kg (99%).
Day 13: Was increased significantly at 3,200 µl/kg.
Day 14: Was decreased significantly (95%) at 3,200 µl/kg and was increased significantly at 1,280 µl/kg (95%).
Day 15: Was decreased significantly (99%) at 3,200 µl/kg.

Dose descriptor:

NOAEL

Effect level:

400 other: mg/kg bw

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

external malformations

skeletal malformations

Abnormalities:

effects observed, treatment-related

Localisation:

external: eye

external: face

external: limb

external: paw

external: tail

skeletal: skull

skeletal: forelimb

skeletal: rib

skeletal: vertebra

skeletal: hindlimb

Description (incidence and severity):

Anophthalmia, exophthalmia, micrognathia, exencephaly, total skull malformation, brachygnathia inferior, spina bifida, kyphosis, cleft palate, oligodactyly, tail shortenings, split of the upper jaw, syndactyly, polydactyly, accessory toes, scoliosis, bent tail, hernia umbilicalis, aplasia of the claw, and ectopia visceralis.

Developmental effects observed:

yes

Lowest effective dose / conc.:

600 other: mg/kg bw

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Pregnant NMRI mice received a single test substance application via gavage at gestation day (GD) 6, 7, 8, 8, 10, 11, 12, 13, 14 or 15. The dose level varied between 400 and 3000 mg/kg bw. The study was terminated at GD18. Maternal and developmental parameters were recorded.

The aim of this study was the determination of the dose level inducing no developmental and maternal effects after single oral application for each GD. The NOAEL varied, mice were most susceptible for developmental effects after single application at GD 9 (embryolethal effects).

The following NOAELs for developmental toxicity were determined:

GD6 1200 mg/kg bw

GD7 1200 mg/kg bw

GD8 600 mg/kg bw

GD9 400 mg/kg bw

GD10 600 mg/kg bw

GD11 600 mg/kg bw

GD12 600 mg/kg bw

GD13 1200 mg/kg bw

GD14 1200 mg/kg bw

GD15 3000 mg/kg bw

Conclusions:

Day 6:
3,200 or 1,280 µl/kg did not induce maternal toxicity.
3,200 µl/kg had an embryo lethal, fetal toxic and teratogenic effect.
1,280 µl/kg did not result in an embryo lethal, fetal toxic or teratogenic effect.

Day 7:
3,200 or 1,280 µl/kg did not induce maternal toxicity.
3,200 µl/kg had a fetal toxic and teratogenic effect. An embryo lethal effect was not observed.
1,280 µl/kg did not result in an embryo lethal or teratogenic effect but a slight fetal toxic effect was observed.

Day 8:
3,200, 1,280 µl/kg, or 640 µl/kg did not induce maternal toxicity.
3,200 µl/kg had an embryo lethal, fetal toxic and strong teratogenic effect.
1,280 µl/kg did not result in an embryo lethal or fetal toxic effect. A strong teratogenic effect was observed.
640 µl/kg did not result in an embryo lethal, fetal toxic or teratogenic effect.

Day 9:
3,200, 1,280 µl/kg, or 640 µl/kg did not induce maternal toxicity.
3,200 µl/kg had an embryo lethal, fetal toxic and teratogenic effect.
1,280 µl/kg did not result in an embryo lethal but fetal toxic and teratogenic effect.
640 µl/kg did not result in an embryo lethal but fetal toxic and teratogenic effect.
427 µl/kg did not result in an embryo lethal, fetal toxic or teratogenic effect.

Day 10:
3,200, 1,280 µl/kg, or 640 µl/kg did not induce maternal toxicity.
3,200 µl/kg had an embryo lethal, fetal toxic and teratogenic effect.
1,280 µl/kg had a weak embryo lethal, fetal toxic and teratogenic effect.
640 µl/kg did not result in an embryo lethal or teratogenic effect but a weak fetal toxic effect.

Day 11:
3,200, 1,280 µl/kg, or 640 µl/kg did not induce maternal toxicity.
3,200 µl/kg had no embryo lethal, but fetal toxic and teratogenic effect.
1,280 µl/kg had no embryo lethal, but fetal toxic and teratogenic effect.
640 µl/kg did not result in an embryo lethal, teratogenic or fetal toxic effect.

Day 12:
3,200, 1,280 µl/kg, or 640 µl/kg did not induce maternal toxicity.
3,200 µl/kg had no embryo lethal, but fetal toxic and teratogenic effect.
1,280 µl/kg had no embryo lethal, but to a questionable fetal toxic and teratogenic effect. The results were not statistically significant, but the type of malformation (oligodactyly) would suggest a treatment-related effect.
640 µl/kg did not result in an embryo lethal, teratogenic or fetal toxic effect.

Day 13:
3,200 or 1,280 µl/kg did not induce maternal toxicity.
3,200 µl/kg had an embryo lethal, weak fetal toxic but no teratogenic effect.
1,280 µl/kg did not result in an embryo lethal, fetal toxic or teratogenic effect.

Day 14:
3,200 or 1,280 µl/kg did not induce maternal toxicity.
3,200 µl/kg had a fragile embryo lethal and fetal toxic effect but no teratogenic effect.
1,280 µl/kg did not result in an embryo lethal, fetal toxic or teratogenic effect.

Day 15:
3,200 or 1,280 µl/kg did not induce maternal toxicity.
3,200 µl/kg had no embryo lethal or teratogenic effect. A weak fetal toxic effect was reported.

Mice were most susceptible for developmental effects after single oral application of the test substance on gestation day 9, the NOAEL for mater and developmental toxicity was 1,200 mg/kg bw and 400 mg/kg bw, respectively.

Executive summary:

The test substance was investigated for its teratogenic potential using pregnant mice after a single oral application on different days (6, 7, 8 and 15 post coitum).

The LD50 calculated was used for the generation of the test concentrations. Once applied orally and after an observation period of 14 days, the LD50 in mice was 6400 µl/kg bw. Test concentrations chosen for the current study were 3,200, 1,280 µl/kg bw, 640 µl/kg bw, and 427 ml/kg bw corresponding to 1/2, 1/5, 1/10, and 1/15 LD50, respectively. Untreated control groups were included.

The oral application of the test substance diluted in destilled water was performed using a cannula on Day 6, 7, 8, 9, 10, 11, 12, 13, 14, or Day 15 post coitum. The application volume was 200 µl.

Caesarean section was performed on Day 18 post coitum. All fetuses were observed for external malformations. Skeletal and visceral malformations as well as variations and retardations were examined on 2/3 and 1/3 fetuses of each litter, respectively.

Doses that did not induce maternal toxicity, fetal toxicity, embryo lethality or a teratogenic effect were analyzed for each day during gestation. In detail, analyzed doses for the individual days were as follows:

Day 6 post coitum (p.c.): 1,280 µl/kg

Day7 p.c.: 1,280 µl/kg +)

Day 8 p.c.: 640 µl/kg

Day 9 p.c.: 427 µl/kg

Day 10 p.c.: 640 µl/kg +)

Day 11 p.c.: 640 µl/kg

Day 12 p.c.: 640 µl/kg

Day 13 p.c.: 1,280 µl/kg

Day 14 p.c.: 1,280 µl/kg

Day 15 p.c.: 3,200 µl/kg +)

+) The application of lower doses was not performed, because only a weak fetal toxic effect was observed.

Mice were most susceptible for developmental effects after single oral application of the test substance on gestation day 9, the NOAEL for mater and developmental toxicity was 1,200 mg/kg bw and 400 mg/kg bw, respectively.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

treatment period gestation day 6-15; partly limited documentation, e.g. no details about the test substance; untreated controls; no historical control data

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

The test was performed according to FDA Guidelines for reproduction studies for safety evaluation of drugs for human use. Food and Drug Administration, Washington, 1966 .

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
- Substance type: technical product
- Substance No.: XX/12
No further details available.

Species:

mouse

Strain:

NMRI

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wiga, Sulzfeld, Germany
- Housing: 5 animals per cage
- Certified diet and tap water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Rel. air humidity: 50-60 %
- Photoperiod: 12 hours dark/12 hours light

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared daily.

Constant application volume in all dose groups: 200 µL per mouse
Dose was related to the initial weight at GD 0.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 2 h
- Proof of pregnancy: [vaginal plug] referred to as day 0 of pregnancy (GD 0).

Duration of treatment / exposure:

Gestation days (GD) 6-15

Frequency of treatment:

Once daily (total application: 10 times)

Duration of test:

Termination at GD 18, caesarean section

Dose / conc.:

256 other: µl/kg bw/day (actual dose received)

Remarks:

corresponding to 240 mg/kg bw/day

Dose / conc.:

427 other: µl/kg bw/day (actual dose received)

Remarks:

corresponding to 400 mg/kg bw/day

Dose / conc.:

1 280 other: µl/kg bw/day (actual dose received)

Remarks:

corresponding to 1200 mg/kg bw/day

No. of animals per sex per dose:

22-24 pregnant animals

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Data from acute oral toxicity in female mice.
- Rationale for animal assignment: Randomisation

Maternal examinations:

CLINICAL SIGNS:
Clinical symptoms were recorded daily.

BODY WEIGHT:
Body weight was measured thrice weekly.

GROSS PATHOLOGY:
A necropsy was conducted at termination.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination.
Examinations included:
- Gravid uterus weight: No data
- Placenta weight of living fetuses: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Dead and living fetuses: Yes

Fetal examinations:

Weight, length and sex of fetuses were determined.

- External examinations: all per litter
- Soft tissue examinations: 1/3 of all fetuses was processed for the assessment of visceral effects (fixation in Bouin's solution for 2 weeks, 15 transversal sections).
- Skeletal examinations: 2/3 of all fetuses was processed for the assessment of skeletal effects (Dawsen-method; Alizarin staining).

Statistics:

Analysis of variance
t-test
Level of significance: p<=0.05

Indices:

See results.

Historical control data:

No data available.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Diarrhea was obseved in two mice treated with 1280 µl/kg bw/day. No toxicity signs were observed in animals treated with 427 and 256 µl/kg bw/day.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1280 µl/kg bw/day, animals showed reduced body weight gain compared to animals of the control. Body weights of animals treated with 427 and 256 µl/kg bw/day showed similar values compared to the control.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects were seen at necropsy.

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

In 16 animals treated with 1280 µl/kg bw/day, an increased amount of amniotic fluid was present in the uterus.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

At 1280 µl/kg bw/day, 41% implantation loss was observed.

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

The dose of 1280 µl/kg bw/day resulted in dead fetuses and embryos during the entire gestation.

Dose descriptor:

NOAEL

Effect level:

400 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

1 200 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Abnormalities:

effects observed, treatment-related

Localisation:

other: dead fetuses, implantation loss

Description (incidence and severity):

41% of implantation loss were observed.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Fetal weight and placental weight were significantly reduced in all treated groups.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

At 1280 µl/kg bw/day, the average number of live fetuses was reduced. Dead implants were increased per percent (99%) in the highest dose group (1280 µl/kg bw/day). All other treated groups were similar to the control.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

External malformations concerning limbs were oligodactyly and syndactyly. Malformations of the head were total or partial exencephaly with partly missig eyelid, brachygnathia inferior and macroglossia. These malformations were observed in the highest dose group (1280 µl/kg bw/day). Total exencephaly and missing eyelid were reported in the 427 µl/kg bw/day group. No effects were observed in the 256 µl/kg bw/day group and control groups with the exemption of one foetus having a bent tail.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Treatment with 1280 µl/kg bw/day resulted in extrem malformations of the fetuses (77.3%), namely at limbs (cleft palate, aplasia of praesphenoid) skull, spine (synostosis of processus spinales, split of vertebral bodies), and ribs (fused ribs). In the corresponding control, only two fetuses (0.87%) developed malformations at the skull (cleft palate).
Cleft palate, total exencephaly, and fused ribs were also noted in 15 (5.95%) fetuses of the 427 µl/kg bw/day group. In the associated control group, only one fetus developed cleft palate.
Cleft palate and fused ribs were noted in three fetuses (1.25%) of the 256 µl/kg bw/day group. In the corresponding control group, 15 fetuses (5.43%) had cleft palate, split of vertebral bodies, and fused ribs. Comapring the lowest dose group (256 µl/kg bw/day) with the corresponding control, the amount of fetuses developing malformations in percent was significantly decreased.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Exencephatly, see "skeletal malformations".

Dose descriptor:

NOAEL

Remarks:

teratogenicity

Effect level:

240 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

240 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: developmental toxicity

Abnormalities:

effects observed, treatment-related

Localisation:

external: limb

skeletal: skull

skeletal: rib

other: skeletal: spine

Developmental effects observed:

yes

Lowest effective dose / conc.:

240 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Developmental toxicity in mice after oral application of N, N-dimethylacetamide

Parameter	Control 1 (untreated)	240 mg/kg bw/day	Control 2 (untreated)	400 mg/kg bw/day	Control 3 (untreated)	1200 mg/kg bw/day
Number of litters	23	22	22	24	23	19
Implants per litter	13.8	13.2	11.1	12.3	12.3	12.6
% dead implants	13.2	17.2	12.9	14.6	19.1	41.0*
No. of living fetuses	276	240	210	252	229	141
No. of male/female fetuses	162/114	152/88	119/91	127/125	121/108	86/55
Living fetuses per litter	12.0	10.9	9.1	10.5	10.0	7.42
Fetal weight in g	1.04 ± 0.15	1.01 ± 0.13*	1.11 ± 0.13	1.03 ± 0.13*	1.17 ± 0.13	0.75 ± 0.14*
Fetal length in cm	2.20 ± 0.15	2.22 ± 0.12	2.25 ± 0.11	2.20 ± 0.14	2.28 ± 0.12	1.91 ± 0.19
Placental weight in g	0.08 ± 0.01	0.07 ± 0.01*	0.08 ± 0.01	0.07 ± 0.01*	0.08 ± 0.01	0.06 ± 0.01*
% fetuses with malformations	5.4	1.2	1.0	5.9	0.9	77.3*
No. of runts	22	23	6	23	3	97

f: females; m: males; *: significant, p<=0.05

Means ± standard deviation given (not available for all parameters)

Conclusions:

Developmental toxicity was found in mice at oral dose levels without maternal toxicity; the LOAEL was 240 mg/kg bw/day, the lowest dose tested. No maternal toxicity was detected at 400 mg/kg bw/day which was set as NOAEL. Teratogenic effects were detected at 400 mg/kg bw/day and above resulting in a NOAEL of 240 mg/kg bw/day.

Executive summary:

The test subtance was investigated for its teratogenic potential using mice received ten times an oral appliaction of either 256, 427, or 1280 µl/kg bw/day. Test concentrations were chosen based on an acute toxicity test that has been conducted prior to this study resulting in a LD50 of about 6400 µl/kg bw. Chosen test concentrations were equivalent to 1/5, 1/15 and 1/25 of the LD50.

Control animals were included and received water. Oral application of the test substance diluted in water was performed with a cannula and an application volume of 200 µl/animal/day was administered from day 6 until day 15 post coitum leading to a total application frequency of ten times.

The operative delivery was performed on day 18 post coitum. All fetuses were observed for external malformations. Two out of three fetuses of one litter each and 1/3 fetus of one litter were observed for skeletal and visceral malformations, variations, and retardation, respectively.

The dose of 1280 µl/kg bw/day resulted in a toxic reaction to maternal mice. Body weight gain was reduced and two animals had diarrhea.

In fetuses, 1280 µl/kg bw/day led to embryo lethality. Embryos and fetuses died during the entire gestation period. Implantation loss were reported to be 41%. Fetuses delivered on day 18 post coitum showed 77% malformations. A reduced body weight and an increased number of skeletal retardations and variatons were regarded as a sign of fetal toxicity.

Maternal animals did not show any signs of toxicity treated with either 427 or 256 µl/kg bw/day. No significant increase in the number of dead fetuses was reported. The number of skeletal and visceral retardations and variations of the fetuses were not correspondent to the dose-independent, however, statistically significant increase of fetal body weight.

Fetuses of the 427 µl/lg bw/day group showed weak signs of teratogenicity. Approximately 6% of live fetuses had malformations. Malformations reported for fetuses in the 256 µl/kg bw/day group were similar to the corresponding control group. No maternal toxicity was detected at 400 mg/kg bw/day which was set as NOAEL. Teratogenic effects were detected at 400 mg/kg bw/day and above resulting in a NOAEL of 240 mg/kg bw/day.

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

10-11 pregnant rabbits per group; exposure restricted to gestation days (GD) 6-18

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

10-11 pregnant rabbits per group; exposure restricted to gestation days (GD) 6-18

Principles of method if other than guideline:

The test was performed according to FDA Guidelines for reproduction studies for safety evaluation of drugs for human use. Food and Drug Administration, Washington, 1966.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
Source: BASF AG
Substance No.: XXIII/236-1
No further details available.

Species:

rabbit

Strain:

other: Russian

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dr. Karl Thomae GmbH, Biberach/Riss, Germany
- Mean body weight at initiation: No data
- Age at initiation: 23-27 weeks old
- Acclimation period: 2 weeks (prior to artificial insemination)
- Housing: Individually
- Certified diet and tap water: ad libitum

ENVIRONMENTAL CONDITIONS:
- Temperature: 20-24 °C
- Humidity: 50-60 %
- Photoperiod: 12 hours dark/12 hours light

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared daily.

The applied dose volume was 10 mL/kg bw.

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- Impregnation procedure: Artificial insemination

Duration of treatment / exposure:

Gestation days (GD) 6-18

Frequency of treatment:

Once daily

Duration of test:

Termination on GD 28

Dose / conc.:

100 other: µl/kg bw/day (actual ingested)

Remarks:

94 mg/kg bw/day

Dose / conc.:

300 other: µl/kg bw/day (actual ingested)

Remarks:

280 mg/kg bw/day

Dose / conc.:

500 other: µl/kg bw/day (actual ingested)

Remarks:

470 mg/kg bw/day

No. of animals per sex per dose:

Initial 12 rabbits per group.
10-11 pregnant rabbits per group were evaluated.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Preliminary experiments (see BASF, 1974 in Section 7.8.2)

Maternal examinations:

CLINICAL SIGNS, MORTALITY:
Clinical signs (mortality) were recorded daily during, prior and after exposure.

BODY WEIGHT:
Body weight was measured thrice weekly and at GD 0, 6, 12, 18, and 28.

FOOD CONSUMPTION:
Food consumption was recorded daily.

GROSS PATHOLOGY:
At termination a necropsy was performed.

OTHER:
The conception rate was assessed. There are no data about water consumption.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination.
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of resorptions (temporally differenciated), dead and living fetuses

Fetal examinations:

Fetal weight, length, sex, viability and placental weight were recorded.

Retardations and variations were recorded as well as malformations, based on:
- External examinations
- Soft tissue examinations
- Skeletal examinations (via x-rays)
- Head examinations (12 transversal sections after fixation in Bouin's solution)

Statistics:

WILLIAMS trend test
FISHER exact test
Mann-Whitney-U test
Level of significance: p<0.05

Indices:

See results

Historical control data:

Yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 470 mg/kg bw/day: Expression of pain and tremor. No effects were observed in the lower treatment groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two rabbits were found dead of the 470 mg/kg bw/day test group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In all treatment groups, significantly reduced body weight gain was observed.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In all treatment groups, significantly reduced food consumption was observed.

Gross pathological findings:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

280 mg/kg bw/day: An increase in implantation loss was observed.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

500 mg/kg bw/day:
A complete resorption of implantations was found.

Other effects:

no effects observed

Description (incidence and severity):

There were no effects on conception rate, corpora lutea, implantation rate, fetal length, and placental weight.

Dose descriptor:

NOAEL

Effect level:

94 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

Dose descriptor:

LOAEL

Effect level:

94 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

increased implantation loss

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

280 mg/kg bw/day: fetal body weight was significantly reduced.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

280 mg/kg bw/day: a slight increase in malformations (5/39 fetuses) were detected (not significant but considered to be of toxicological relevance). Malformations observed were cleft palate (4/5) and fused ribs (1/5). Two out of five fetuses with malformations showed two alterations; one fetus showed cleft palate and oedema in throat area, one further fetus showed fused ribs and microphthalmia unilateral. The 5 malformed fetuses were observed in three litters resulting in 33.33 percent which was not significant. Variations and retardations were observed in 33/39 living fetuses across all litters resulting in 86.11%.

Dose descriptor:

NOAEL

Effect level:

94 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

Abnormalities:

effects observed, treatment-related

Localisation:

external: face

external: thorax

Description (incidence and severity):

a slight increase in malformations (5/39 fetuses) were detected. Malformations observed were cleft palate (4/5) and fused ribs (1/5).

Developmental effects observed:

yes

Lowest effective dose / conc.:

280 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Developmental toxicity in rabbits after oral application of N,N-dimethylacetamide

Parameter	Untreated control	94 mg/kg bw/day	280 mg/kg bw/day	470 mg/kg bw/day
Pregnant rabbits	10/12	11/11	10/10	11/12
Corpora lutea/dam	7.8	8.5	7.9	Not applicable
Implatations/dam	6.4	6.8	6.5	6.8
% Living fetuses per implantations per dam	83.3	88.2	64.6	Complete implantation loss
Total dead fetuses	2	-	-	-
% resorptions per pregnant dam	16.7	11.8	35.4 (toxicological relevance)	100
Fetal weight m&f (g)	32.9 ± 5.2	32.2 ± 3.3	26.1 ± 5.0*	Not applicable
Total runts	0	1	0	Not applicable
No. of malformed fetuses	0/54	0/65	5/39	Not applicable
Litters with malformed fetuses	0	0	3	Not applicable
% living fetuses/litter with variations and retardations	83.7	65.7	86.1	Not applicable

Means ± standard deviation

*: p<0.05

Conclusions:

The NOAEL for developmental effects (maternal and offspring) in rabbits after oral exposure was 94 mg/kg bw/day and the LOAEL was 280 mg/kg bw/day; maternal toxicity was found at 94 mg/kg bw/day (LOAEL).

Executive summary:

The test substance was investigated for its prenatal toxicity in rabbits after oral application of thirteen times. Investigation have been conducted in October 1975 and from January until March 1976 according to guidelines set forth by the FDA (1966). Animals were exposed to 100, 300, and 500 µl/kg bw. The test substance was diluted in bidistilled water and applied via cannula to 10 ml/day from Day 6 to Day 18 post insemination. All animals were sacrified on Day 28 post insemination. All fetuses were observed for external malformations and received two-plane x-ray. Skelteal evaluations were based on x-ray results. The heads of the fetuses were fixed in Bouin'scher solution. Using the method proposed by Wilson (1965), approximately 3 -4 weeks after fixation, ca. 8 -10 transverse cross sections were made and evaluated from each head.

100 µl/kg bw of the test substance were well tolerated by animals without any signs of toxicity. Reduced body weight gain correlated with a reduced food consumption. There were no unscheduled deaths. The number of corpora lutea, implantations, and living fetuses were comparable to control animals. No adverse effects were noted when animals were examined macroscopically. Parameters observed in fetuses (body weight, lenght, weight of placenta, number and types of variations and retardations) were similar to those observed in control animlas. No malformations were reported in fetuses.

300 µl/kg bw of the test substance were tolerated by animals without any signs of toxicity. Reduced body weight gain correlated with a reduced food consumption in a dose-dependant manner. The number of corpora lutea and implantations were comparable to control animals. The number of living fetuses was reduced and the number of resorptions was increased. Based on recommended number of litters (FDA guideline), the difference was not significant. No adverse effects to the inner organs were reported when animals were examined macroscopically. The average body weight of fetuses was reduced, however, lenght, weight of placenta, and number and types of variations and retardations were normal. No malformations were reported in fetuses.

Animals treated with 500 µl/kg bw of the test substance experienced pain, tremor, and reduced feces as a consequence of an adverse reduction in food consumption. Body weight gain was reduced. One animal died after the eighth and thirteenth application. As a result of early dead fetuses or rather both of the maternal animals, counting of copora lutea was inaccurate, hence, an evaluation was not performed. Conception rate and number of implantations were comparable to control animals. All emryos were resorbed. A defined lobule was investigated in one maternal animals after section. All other examined animals did not show any macroscopic alterations.

Under the conditions chosen, the NOAEL for developmental effects in maternal animals and in the fetus was concluded to be 94 mg/kg bw/day.

Reference 6

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jul 1973 - Mar 1974

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

low number of dams, exposure restricted to gestation days (GD) 6-18

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

low number of dams, exposure restricted to gestation days (GD) 6-18

Principles of method if other than guideline:

According to "Guidelines for reproduction studies for safety evaluation of drugs for human use" by FDA (1966) and "Principles for the testing of drugs for teratogenicity"; WHO 1967.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
No details available.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Laboratorium Pharmakologie und Toxikologie, Hamburg, Germany
- Age at study initiation: 8-9 months
- Weight at study initiation: 3.5-4.4 kg
- Housing: individually
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 0.5
- Humidity (%): 60 +/- 3
- Photoperiod (hrs dark / hrs light): 12/12 (intensity: 300 lux)

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on exposure:

VEHICLE:
Controls received 330 µL/kg bw/day 1 % carboxyethyl cellulose in water

Analytical verification of doses or concentrations:

no

Details on mating procedure:

- M/F ratio per cage: 1/1
- Proof of pregnancy: successful mating = GD 0

Duration of treatment / exposure:

Gestation days (GD) 6-18

Frequency of treatment:

Once daily

Duration of test:

Termination on GD 29

Dose / conc.:

100 other: µl/kg bw (actual ingested)

Remarks:

corresponding to 94 mg/kg bw/day

Dose / conc.:

300 other: µl/kg bw (actual ingested)

Remarks:

corresponding to 280 mg/kg bw/day

Dose / conc.:

900 other: µl/kg bw (actual ingested)

Remarks:

corresponding to 850 mg/kg bw/day

No. of animals per sex per dose:

10 pregnant rabbits

Control animals:

yes, sham-exposed

Details on study design:

The day of copulation was designated as Day 0.

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Once daily (every morning at the same time)

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: not specified

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: uterus
- Gross pathology at necropsy

Ovaries and uterine content:

The ovaries and uterine content was examined after termination.
Examinations included:
- Gravid uterus weight,
- Number of corpora lutea
- Number of implantations
- Number of early and late resorptions

Fetal examinations:

- number of fetuses
- sex and viability was determined. The fetus was viable when living was confirmed after a 6- or 24-h-incubation (37°C). Living was charaterized by breathing and movement.
- number and size of resorptions
- corpora lutea and position of the fetus in the uterus
- body weight, including runts
- external malformations
- variations and retardations
- gross pathology
- the skeletal system was dyed with alizarine and examined with the method proposed by Dawson

Statistics:

student t-test with significance p < 0.01

Indices:

resorption rate, malformation rate, variation rate, pre-implantation loss, post-implantation loss

Historical control data:

Available

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 0.9 ml/kg, animals showed sedation and ataxia from day 2 onwards. The symptoms worsened quickly. From day 4-6, diarrhea with mushy, mucous faeces was observed. The only effect observed in animals treated with lower doses was a loss of appetite during the first four days of treatment in the 0.3 ml/kg group.

Mortality:

mortality observed, treatment-related

Description (incidence):

All ten animals from the highest dose group (0.9 ml/kg) died within 5-8 days after a coma of several hours after commencement of treatment. In four cases, slight agonal cramps were observed before death. No mortality was observed in the lower dose groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A reduction in body weight was observed in the highest treatment group (0.9 ml/kg).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was reduced in the 0.9 ml/kg group.

Water consumption and compound intake (if drinking water study):

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 0.9 ml/kg, animals showed pale, yellowish parenchymatous organs, pronounced hepatic lobules, and a mushy mass found in the large (partially also in the small intestine) intestine infused with a moderate amount of light-red blood.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

At 0.3 ml/kg, postimplantation loss was 45.4%.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 0.3 ml/kg, resorption rate was increased (not significant due to the high standard deviation)

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

Eight dead fetuses were found in the 0.3 ml/kg group.

Dose descriptor:

NOAEL

Effect level:

280 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

mortality

Dose descriptor:

NOAEL

Effect level:

94 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

dead fetuses

early or late resorptions

pre and post implantation loss

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

postimplantation loss, increased resorption rate, dead fetuses

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weight at 0.3 ml/kg.

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

At 0.3 ml/kg, number of fetuses was reduced (not significant due to the high standard deviation).

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Slight increase in the rate of malformations (0.41 % in historical controls, 0 % in concurrent control and 4.9 % in treated dams) were noted in the 0.3 ml/kg group. Three out of 61 fetuses were malformed; cleft palate, a cyst in the cortex of the right kidney or exencepahly were reported.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Cyst in the cortex of the right kidney (see external malformations).

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Increased variations (mainly retardations) at 0.3 ml/kg.

Dose descriptor:

NOAEL

Effect level:

94 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

external malformations

other: increased variations (mainly retardations)

Abnormalities:

effects observed, treatment-related

Localisation:

external: face

skeletal: skull

visceral/soft tissue: urinary

Description (incidence and severity):

cleft palate, cyst in renal cortex

Developmental effects observed:

yes

Lowest effective dose / conc.:

280 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Conclusions:

Developmental toxicity was found in rabbits at oral dose levels without maternal toxicity. The NOAEL for maternal toxicity was 280 mg/kg bw/day, the NOAEL for developmental toxicity was 94 mg/kg bw/day.

Executive summary:

The test substance was examined for its effects on pregnant rabbits and the fetuses. Thirty animals were used in the treatment groups and ten untreated animals served as the control group. Test concentrations were chosen to be 0.1, 0.3, and 0.9 ml/kg bw. The undiluted test substance was administered orally via gavage from Day 6 through Day 28 of the gestation period. Control animals received 0.33 ml of a 1% carboxyethyl cellulose gel/kg bw by gavage.

The lowest treatment group (0.1 ml/kg bw) did not reveal any signs of toxicity to maternal animals or fetuses. All results were comparable to control group.

At 0.3 ml/kg bw, maternal animals suffered from a temporary loss of appetite. Number of living offspring was reduced. A higher resorption rate and a post-implantation loss of 45.5% (control: 11.5%) were observed. Body weight of fetuses was slightly reduced and the rate of variations enhanced. It is suggested that at a dose level of 0.3 ml/kg bw, the teratogenic potential of the test substance commences characterized by one fetus with cleft palate, a cyst in the cortex of the right kidney or an exencephaly.

The highest dose (0.9 ml/kg bw) was in the LD100 range; all animals died until Day 8 of sedation, ataxia, poor appetite, reduced body weight, diarrhea, coma of several hours, and occasional slight agonal cramps. At necropsy, pale parenchymatous organs, pronounced hepatic lobules, and a mushy, haemorrhagic imbibed mass in the large intestine (partially also in the small intestine) were found. A differentiation whether fetuses were already resorbed or were viable on the day of death could not be performed.

Under the conditions chosen, the lowest toxic dose for maternal animals and fetuses via gavage is suggested to be around 0.3 ml and between 0.1 -0.3 ml, respectively.

Reference 7

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

minor restriction: no data about impurities of the test substance

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
- Purity: ≥99 %
- Source: Haskell No.: 22203
No further details available.

Species:

rat

Strain:

other: Crl :CD®(SD)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Age at arrival: 64 days old
- Body weight: 174-228 g
- Housing: Individually
- Diet (certified) and tap water: ad libitum (periodically analysed for contaminations)
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22-24 °C
- Humidity: 40-60 %
- Photoperiod: 12 hours dark/12 hours light

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
HPLC-grade water was used; the dose volume was 10 mL/kg bw.
Solutions of the test substance in the vehicle were prepared weekly. The volume administered was based on the most recent body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from three batches of test solutions were collected. Analyses of all samples addressed concentration. The homogeneity and stability of DMAC dosing formulations was demonstrated. GC methods; stock solution of the analytical standard (DMAC, 99.9 % pure) used for calibration standards. Analysis showed that DMAC was at targeted concentrations (±8 % of nominal).

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1/1
- Proof of pregnancy: Vaginal plug referred to as day 1 of pregnancy.

Females were cohabited with males (1:1) until copulation was confirmed by the presence of a copulation plug in the vagina or on the cageboard. The day copulation was confirmed was designated day 1 of gestation (GD 1)

Duration of treatment / exposure:

Gestation days (GD) 7-21

Frequency of treatment:

Once daily

Duration of test:

On GD 22, all female rats were sacrificed and necropsied.

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Dose / conc.:

65 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 mated females; 24-25 litters

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection via a pilot study:
8 mated rats were gavaged at GD 7-21 at daily dose levels of 0, 62.5, 125, 250, or 500 mg/kg bw/day; termination on GD 22.

Results:
500 mg/kg bw/day: Maternal body weight gain and food consumption significantly reduced, increased liver and kidney weight in dams and increased clinical chemistry parameters, red-coloured vaginal discharge; increased resorptions, only 5 viable fetuses, malformations.
250 mg/kg bw/day: Maternal body weight gain significantly reduced, increased liver and kidney weight, increased clinical chemistry parameters; decreased fetal weight, 3 fetuses with malformations.
125 mg/kg bw/day: Increased liver and kidney weight, and increased clinical chemistry parameters; 3 fetuses with cleft palate.
65 mg/kg bw/day: Significantly increased liver weights in dams.

Randomisation main study:
Females were ranked by their body weights on GD 1 and randomly assigned to control or experimental groups.

Maternal examinations:

CLINICAL SIGNS:
Clinical signs were recorded twice daily.

BODY WEIGHT:
Prior to the start of dosing, females were weighed; during the study, females were weighed on GD 1, 7-22;

FOOD CONSUMPTION:
Food was weighed on GD 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 22.

CLINICAL CHEMISTRY:
Blood was sampled prior to sacrifice. The following parameters were checked.
AIkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), sorbitol dehydrogenase (SDH), bilirubin (BILRN), cholesterol (CHOL), triglycerides (TRIG), total protein (TPROT), albumin (ALBMN), globulin (GLOBN), glucose (GLUCO), urea nitrogen (BUN), creatinine (CREAT), phosphate (PHOS), calcium (CALC), sodium (Na), potassium (K), chloride (Cl)

GROSS PATHOLOGY:
A complete necropsy was performed at termination

ORGAN WEIGHTS:
Liver and kidneys were weighed.

HISTOPATHOLOGY:
A histopathological examination of liver and kidneys was conducted.

Ovaries and uterine content:

The uterus was removed, weighed, and opened; live and dead fetuses, and resorptions were counted and their relative positions were recorded; empty uterus was weighed; ovaries removed and the corpora lutea counted; uterus of each non-pregnant rat was opened and stained with ammonium sulfide to detect very early resorptions, data collected from those animals were used only to determine the incidence of pregnancy and the number of females with total resorptions.

Fetal examinations:

Live fetuses were weighed, sexed, and examined for external alterations; first live fetus and thereafter every other fetus in each litter was decapitated and examined for visceral alterations and the sex verified; retarded renal development was classified using the scheme of Woo and Hoar; heads were fixed in Bouin's fluid and examined; remaining fetuses were euthanized by an intraperitoneal injection of sodium pentobarbital; all fetuses were fixed in 70 % ethanol, eviscerated (if not done earlier during the visceral examination), macerated in 1 % aqueous potassium hydroxide solution, stained with alizarin red, and examined for skeletal alterations.

Statistics:

Linear contrast of means
Jonckheere's test
Cochran-Armitage test
Linear contrast of least square means
Several tests for clinical chemistry
Level of significance: p<=0.05

Historical control data:

Yes, compiled by the Middle-Atlantic Reproduction and Teratology Association (MARTA).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Non-pregnant animals were excluded. Significantly reduced maternal body weight gain was noted at 400 mg/kg bw/day at GD 7-9, 9-11, 15-17, 17-19, 19-21; at 150 mg/kg bw/day maternal weight changes were reduced at GD 7-9, and significantly reduced at GD 9-11; no dose-related effects on maternal weight changes were seen at either 65 or 20 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 400 mg/kg bw/day there was significantly reduced food consumption over GD 7-9, 9-11, 11-13, 15-17, 19-21.

Clinical biochemistry findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant increases in mean absolute kidney weight and in mean relative liver and kidney weights in the 400 mg/kg dose group were found; and considered to be test substance-related but not biologically adverse since absolute weight increase was minimal, no histopathological effects were detected, and clinical chemistry data were normal (effects adaptive response during exposure to DMAC).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects but placentas from 14 of 25 dams had white or tan outer edges at 400 mg/kg bw/day (consistent with the advers maternal and developmental toxicity).

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Increased mitotic figures were noted in livers of dams at 400 mg/kg bw/day; this finding was considered not to be adverse since no degeneration or inflammation was found and clinical chemistry was normal (associated with metabolism of the test substance).

Other effects:

effects observed, treatment-related

Description (incidence and severity):

At 400 mg/kg, the placentas from 14 of 25 dams had white or tan outer edges. This observation is most likely related to and consistent with the adverse maternal and developmental toxicity seen at this dose level. This observation was also reported for one, zero, two, and two animals from the 0, 20, 65, and 150 mg/kg groups, respectively. There were no other remarkable postmortem findings.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test substance-related effects concerning dams with the mean number of implantations.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no test substance-related effects concerning dams with total resorptions.

Description (incidence and severity):

Effects on pregnancy duration: No effects observed.
Description (incidence and severity): There were no test substance-related effects concerning dams that delivered early.

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

65 mg/kg bw/day (actual dose received)

Basis for effect level:

body weight and weight gain

Dose descriptor:

LOAEL

Remarks:

maternal toxicity

Effect level:

150 mg/kg bw/day

Basis for effect level:

body weight and weight gain

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significant reduced fetal body weight was noted at 150 and 400 mg/kg bw/day

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

Significantly reduced number of live fetuses at 400 mg/kg bw/day.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There were no test substance-related effects concerning mean litter sex ratio.

Changes in postnatal survival:

effects observed, treatment-related

Description (incidence and severity):

Decreased embryofetal viability, and early and late resorptions were significantly increased at 400 mg/kg bw/day.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

There were four affected fetuses from one litter with distended brain ventricles (toxicological relevance unclear), naris atresia, cleft palate, heart and vessel malformations, macroglosia, micrognathia, and synotia at 150 mg/kg bw/day. At 400 mg/kg bw/day, incidence of fetal malformations was significantly increased (synotia, anasarca, micrognathia, naris atresia, malformations of the heart and great heart vessels, distended lateral brain ventricles, fused ribs, absent vertebrae, and hemivertebrae).

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

At 400 mg/kg bw/day, incidence of fetal variations was significantly increased (patent ductus arteriosus and delayed sternebral ossification).

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

65 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

external malformations

visceral malformations

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in postnatal survival

external malformations

visceral malformations

Abnormalities:

effects observed, treatment-related

Localisation:

external: face

visceral/soft tissue: cardiovascular

Developmental effects observed:

yes

Lowest effective dose / conc.:

150 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Developmental toxicity in rats after oral exposure via gavage at gestation days 7-21

Dose in mg/kg bw/day

Parameter	0	20	65	150	400
Early resorption/litter	0.4	0.6	0.6	0.5	3.1*
Late resorptions/litter	0	0	0	0	0.3*
Live fetuses /litter	14.1	13.6	14.1	14.5	10.4*
Mean fetal weight	4.88	5.03	4.99	4.69*	3.22*

*: p<=0.05

Conclusions:

In a gavage study in rats maternal and developmental toxicity was seen after treatment with the test substance at a dose level of 150 mg/kg bw/day, the NOAEL was 65 mg/kg bw/day.

Executive summary:

The test substance diluted in water was administered to groups of 25 mated rats over days 7-21 of gestation (days 7-21G) at daily dose levels of 0, 20, 65, 150, or 400 mg/kg. On day 22G, all rats were euthanized and grossly necropsied . Near the end of the study, blood was drawn and evaluated for a battery of clinical chemical parameters. At necropsy, samples of liver and kidney tissue were taken and evaluated histopathologically . The fetuses were removed from the uterus and were weighed, sexed, and examined for external, visceral, head, and skeletal alterations. Significant, adverse maternal and developmental toxicity were produced at 400 mg/kg . Minimal maternal and developmental toxicity were seen at 150 mg/kg.

At 400 mg/kg, maternal toxicity was evident as significant, dose-related reductions in mean maternal body weight, weight change, and food consumption . No dose-related, adverse effects on any clinical chemistry parameter were seen at any dose level nor were there any adverse histopathological changes seen in either the liver or the kidney. Developmental toxicity was evident as significantly increased embryolethality (increased resorptions), malformations (synotia, anasarca, micrognathia, naris atresia, malformations of the heart and great heart vessels, distended lateral brain ventricles, fused ribs, absent vertebrae, hemivertebrae), and variations. Mean fetal weight was also significantly decreased.

At 150 mg/kg, maternal toxicity was evident as a significant, dose-related reduction in maternal weight gain at the onset of the dosing period . Developmental toxicity was evident as a significant decrease in mean fetal weight . In addition, at 150 mg/kg, there was one fetus with malformations which included naris atresia, heart and vessel malformations, cleft palate, macroglossia, micrognathia, and synotia . Despite the fact that

there was only one affected fetus, this may represent the bottom end of the dose response curve for malformations given the similarity of specific malformations relative to those seen at 400 mg/kg.

There was no evidence of either maternal or developmental toxicity at 65 or 20 mg/kg .

Reference 8

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

TEST MATERIAL
- Source: H. C. Ryall, Belle, West Virginia
- Physical appearance: colorless liquid
- Purity: 99.9%
- Impurities: acetic acid (<100 ppm), methylamine (<2 ppm) and water (<300 ppm)
- Physical state: liquid

No further details available.

Species:

rat

Strain:

other: Crl:CD(SD)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: about 90 days of age (males), about 60 days of age (females),
- Weight at study initiation: 165.0-190.4 g
- Fasting period before study: No
- Housing: Singly in stainless steel, wire-mesh cages throughout the study.
- Diet: Rodent Chow ad libitum, except during exposure
- Water: ad libitum, except during exposure
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 71.9-77.8 °F (except for three days with 80-82 °F during the treatment period)
- Humidity: 28-50 %
- Air changes: 12 air changes per hour
- Photoperiod: 12 hours dark/12 hours light

In Life Dates: From November 22, 1982 to December 13, 1982.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: nitrogen stream, diluted with room air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION / EXPOSURE
Groups of 25 pregnant rats were exposed at target concentrations of 0, 30, 100, or 300 ppm test substance. Rats were exposed by whole-body inhalation for 6 h per day from gestion days (GD) 6-15 (10 exposures). Exposures were conducted in Rochester-type chambers made of stainless steel. The control and the two lowest exposure groups were tested in 750 L chambers, but the high exposure group was tested in a 900 L chamber. A maximum of 55 rats were present in each of the 750 L chambers; 85 were present in the 900 L chamber. Chamber temperature and oxygen concentration was monitored continuously during the exposure period. The control dams were exposed to air only. Liquid test substance was slowly dripped through a preheated (200-225 °C) inclined tube furnace using a syringe drive. Nitrogen circulating at 1-2 L/min carried the vapours through the tube. Non-volatilized material was collected in a trap as it left the furnace. The test substance vapour stream was mixed with dilution air at about 500 L/min and then it was introduced tangentially into the top of the chamber. Non-volatilized material was collected in a trap at the exit of the furnace. Atmospheres were vented through the bottom of each chamber and passed through an aqueous scrubbing tower to remove the test substance from the exhaust air.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance concentrations were analyzed using a gas chromatograph (GC) with a flame ionization detector. Standards were prepared by quantitative dilution of the test substance in acetone. The GC was calibrated daily by injection of liquid standards. Test chambers were analyzed at 30-min intervals by drawing atmospheres through two glass impingers containing acetone, and connected in series.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1/1
- Proof of pregnancy: Vaginal plug referred to as day 1 of pregnancy.

Animals were bred from November 22-26, 1982. The female rats were cohabited overnight with mature males (1 rat/sex/cage). Mating was verified each morning by detecting a copulation plug in the vagina or on the cage board. The day a plug was found was designated Day 1 of gestation (GD 1). Mated females were stratified by body weight and assigned to groups by random sampling within each stratum (25 per group).

Duration of treatment / exposure:

Gestation days (GD) 6-15./exposure of 0, 30, 100, or 300 ppm

Frequency of treatment:

Daily (6 hours/day)

Duration of test:

10 days of exposure, termination on GD 21

Dose / conc.:

32 ppm (analytical)

Remarks:

Value expressed as mean ± 3.9 (SD). 30 ppm (nominal).

Dose / conc.:

100 ppm (analytical)

Remarks:

Value expressed as mean ± 11.4 (SD). 100 ppm (nominal).

Dose / conc.:

281 ppm (analytical)

Remarks:

Value expressed as mean ± 29.4 (SD). 300 ppm (nominal).

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: A pilot study was conducted to generate data necessary for selection of exposure levels for the main study. Groups of 4 dams were exposed by inhalation to 0, 100, 300, or 625 ppm test substance for 6 h per day from days 6 through 15 of gestation (GD 6-15). At the highest exposure level, dams lost weight and only one fetus survived to sacrifice. A decrease in body weight gain among dams exposed to 300 ppm was noted between GD 6-15. No adverse effects were demonstrated among dams exposed to 100 ppm or fetuses exposed to 100 or 300 ppm.

- Rationale for animal assignment: Pregnant females were stratified by body weight and assigned to groups by random sampling within each stratum.

Maternal examinations:

CAGE SIDE OBSERVATIONS:
Clinical signs were recorded (no further details).

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Individual clinical observations were conducted upon arrival, before breeding, each morning from day 1-21 of gestation (GD 1-21) and each afternoon from GD 6-15 (exposure period). Group clinical observations were conducted at the midpoint of each daily exposure period.

BODY WEIGHT:
- Time schedule: Upon arrival, just before breeding and on GD 1, 6, 9, 13, 16 and 21.

POST-MORTEM EXAMINATIONS:
- Sacrifice on GD 21
- Organs examined: Liver was removed and weighed, gravid and empty uterus was weighed.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination.
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: All per litter
- Soft tissue examinations: All per litter
- Skeletal examination: All per litter
- Head examinations: All per litter

Statistics:

See Table 1.

Indices:

See results

Historical control data:

No data

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Stained fur on the crown of the head of some dams was the only clinical sign noted from exposure. This was not different from control animals. No adverse clinical signs were noted.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Dams experienced a significant decrease in body weight gain observed on Days 6-8 and 6-15 at the 300 ppm level. No adverse effect was detected at the 30 or 100 ppm levels.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A bladder containing calculi from a dam exposed to 100 ppm was the only adverse clinical sign noted when maternal viscera were examined at sacrifice.

Details on maternal toxic effects:

Maternal toxicity at the 300 ppm level was demonstrated by a significant decrease in body weight gain observed on Days 6 through 8G and 6 through 15G. Maternal body weight was not adversely affected at the 30 or 100 ppm levels. No adverse effect that was concentration-related was noted for any reproductive parameter.

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

A significant decrease in fetal body weight that was concentration related was demonstrated between the control and 300 ppm groups

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There was no significant difference between control and experimental groups in the total number of developmental variations or variations due to retarded development.

Details on embryotoxic / teratogenic effects:

Exposure to 30 or 100 ppm had no adverse effect on fetal development.

Dose descriptor:

NOAEL

Effect level:

100 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: fetotoxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 2
Summary of Fetal Development - Mean fetal body weight (grams)
0.0 ppm	30 ppm	100 ppm	300 ppm
3.5 (± 0.05)	3.5 (± 0.06)	3.5 (± 0.06)	3.3(± 0.05)*
Stunted fetuses were excluded. Significant exposure-related response detected by Jonckheere’s test, p≤0.05. * Significantly different from control value by two-tailed Mann-Whitney U test, p≤0.05.

Conclusions:

Maternal and fetal toxicity were noted at the 300 ppm exposure level. No adverse effects to the dam or fetus resulted from inhalation exposures to the test substance at 30 or 100 ppm. The test substance was not teratogenic even at a level that was maternally toxic. The study and conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).

Executive summary:

Groups of 25 pregnant rats (100 in total) were exposed by inhalation to nominal concentrations of 0, 30, 100, or 300 ppm of the test substance for 6 hr/day from Days 6 through 15 of gestation. The actual concentrations generated were 0, 32, 100, and 281 ppm of the test substance. The control group received room air.

At the 300 ppm level, significant (p<0.05) decreases in maternal body weight gain were noted during Days 6-8 and Days 6-15 of gestation. No adverse effects were observed among dams exposed to 30 or 100 ppm. Fetal toxicity at 300 ppm was demonstrated by a significant (p<0.05) decrease in body weight. No adverse effects were noted among fetuses exposed to 30 or 100 ppm. The frequency of malformations was similar between the control and experimental groups.

Therefore, maternal and embryo-fetal toxicity was noted at 300 ppm. An apparent no-effect level for both dam and conceptus was 100 ppm. The test substance was not teratogenic even at a level that was maternally toxic.

Reference 9

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

viral infection of rats in all groups hampered evaluation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
- Purity 99.72 %
- Source: Monsanto
No further details available.

Species:

rat

Strain:

other: COBS CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age: 12 weeks old at gestation day (GD) 0

No further details available.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Dose volume: 10 mL/kg bw

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1/1
- Proof of pregnancy: Vaginal plug referred to as day 0 of pregnancy.

Duration of treatment / exposure:

GD 6-19

Frequency of treatment:

Once daily

Duration of test:

GD 20, dams sacrificed

Dose / conc.:

65 mg/kg bw/day (actual dose received)

Dose / conc.:

160 mg/kg bw/day (actual dose received)

Dose / conc.:

400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 rats

Control animals:

yes, concurrent vehicle

Details on study design:

No further details available.

Maternal examinations:

CLINICAL SIGNS:
Clinical signs were recorded once daily.

BODY WEIGHT:
Maternal body weight was recorded at GD 0, 6, 9, 12, 16, and 20.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination.
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:

- Fetal weight and sex
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: No data

Statistics:

Yes

Indices:

No data

Historical control data:

No data

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs due to the treatment were observed (but viral infection).

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A significant reduction in maternal body weight at 400 mg/kg bw/day was observed. A slight reduction in body weight gain was observed in the 0- to 20-day interval adjusted weights in the 160 mg/kg/day group. No effects were seen in the 65 mg/kg/day group.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

There was a significant increase in postimplantation loss per dam at the 400 mg/kg/day level when compared to control.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

At 400 mg/kg/day, high incidence of early resorptions and an increase in the number of late resorptions were noted. One female in the 65 mg/kg/day group and two females in the 160 mg/kg/day group had resorptions.

Dead fetuses:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No biologically meaningful or statistically significant differences in the mean number of corpora lutea.

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Dose descriptor:

NOAEL

Effect level:

160 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

early or late resorptions

pre and post implantation loss

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

postimplantation loss increased, high incidence of early resorptions.

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean fetal body weight was decreased in the 160 and 400 mg/kg/day groups in a dose-response related pattern and was statistically significant at 400 mg/kg/day level. Fetal body weights at the 65 mg/kg/day group were similar to the control group.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

External malformations:

effects observed, treatment-related

Description (incidence and severity):

At 400 mg/kg/day, heart and/or vessel malformations (33 fetuses, 18 litters) were observed and statistically significant. Other malformations occurring in more than one litter included cleft palate (3 fetuses, 3 litters), and anasarca (5 fetuses, 2 litters). The majority of the other malformations occurred in single instances.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Increased skeletal variations included unossified aternebrae Nos. 5 and 6, unossified sternebrae Nos. 1-4, and reduced ossification of skull and vertebrae. Other variations included 25 presacral vertebrae, 14th rudimentary or 14th full ribs, and major vessel variations in the 400 mg/kg/day group. At 160 mg/kg/day, the 25 presacral vertebrae were also increased when compared to historical control data.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Truncus arteriosis, no ductus arteriosis, interventricular septal defects

Dose descriptor:

NOAEL

Effect level:

65 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Abnormalities:

effects observed, treatment-related

Localisation:

external: face

skeletal: skull

skeletal: rib

skeletal: vertebra

visceral/soft tissue: cardiovascular

other: skeletal: sternebrae

Developmental effects observed:

yes

Lowest effective dose / conc.:

160 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

no

Conclusions:

There was maternal toxicity in rats after gavage of the test substance at 400 mg/kg bw/day, NOAEL 160 mg/kg bw/day. The NOAEL for developmental toxicity in fetuses was 65 mg/kg/day.

Executive summary:

The test substance was investigated for its teratogenic potential using 25 pregnant CD rats per group. The test substance was diluted in deionized water and applied once daily by gavage starting with Day 6 of gestation through Day 19. Concentrations used were 65, 160, and 400 mg/kg/day. Cesarean sections were conducted on all maternal animals on gestation Day 20. No treatment-related effects were observed in mortality, appearance, or behavior at necropsy. At 400 mg/kg/day only, mean maternal body weight gain was significantly reduced and increased implantation loss was observed manifesting fetal toxicity. Reduction in mean fetal body weights was reported at 160 and 400 mg/kg/day test groups corresponding to increased developmental variations (reduced ossification and unossified skeletal variations) observed at 400 mg/kg/day dose level. In addition, malformations of the heart, oral cavity and major vessels, and anasarca were seen at 400 mg/kg/day dose level and were regarded as treatment-related. No teratogenic effects were reported at or below dosage level of 160 mg/kg/day.

Reference 10

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

only 10 dams per dose level; no data about vapour generation; some parameters of developmental toxicity not recorded

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
- Purity: >99.9 %
- Source: Wako Pure Chemical Industries, Ltd. (Osaka).
- Stability: Analysed by infrared and mass spectroscopy for purity and for stability by GC, no decomposition or impurities.
No further details available.

Species:

rat

Strain:

other: Crj:CD(SD)IGS

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan
- Age at mating: 10 weeks
- Weight at study initiation: No data
- Fasting period before study: No data
- Housing: Individually
- Certified diet and tap water: ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 21-25 °C
- Humidity: 35-75 %
- Air changes: No data
- Photoperiod: 12 hours dark/12 hours light

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

not specified

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Data on generation of vapour not given (further citation).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Every 15 minutes vapour concentration was measured by a GC method.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy.

Duration of treatment / exposure:

Gestation days (GD) 6-19

Frequency of treatment:

6 h/day

Duration of test:

GD 20 (termination)

Dose / conc.:

102 ppm (analytical)

Remarks:

Value expressed as mean ± 2.7 (SD). 100 ppm (nominal). Equivalent to 360 mg/m³.

Dose / conc.:

298 ppm (analytical)

Remarks:

Value expressed as mean ± 4.0 (SD). 300 ppm (nominal). Equivalent to 1080 mg/m³.

Dose / conc.:

447 ppm (analytical)

Remarks:

Value expressed as mean ± 5.0 (SD). 450 ppm (nominal). Equivalent to 1620 mg/m³.

Dose / conc.:

594 ppm (analytical)

Remarks:

Value expressed as mean ± 6-5 (SD). 600 ppm (nominal). Equivalent to 2160 mg/m³.

No. of animals per sex per dose:

10 pregnant rats

Control animals:

yes, sham-exposed

Details on study design:

No further details available.

Maternal examinations:

CLINICAL SIGNS:
Clinical signs were recorded once daily.

BODY WEIGHT:
Body weight was measured on GD 6, 7, 9, 13, 17, and 20.

CLINICAL CHEMISTRY:
Blood sampling was performed on GD 20. AST, ALT and LDH activities were determined.

GROSS PATHOLOGY:
A necropsy was conducted on GD 20

ORGAN WEIGHTS
Liver weight was recorded.

HISTOPATHOLOGY:
Liver histopathology was performed.

Ovaries and uterine content:

Numbers of resorptions and implantations were determined, no further parameters (e.g. uterus weight and corpora lutea) were assessed.

Fetal examinations:

Numbers of live and dead fetuses, and fetal weight and sex were determined.

Fetuses were examined for malformations/variations
- External examinations: All fetuses
- Soft tissue examinations: Half of fetuses (Bouins fixation)
- Skeletal examinations: Half of fetuses (fixed in 99.5 % ethanol, eviscerated, macerated in 1.5 % KOH solution, and stained with Alizarin red S)
- Head examinations: No data

Statistics:

Dunnett's test
Bartlett's test
One way ANOVA
Kruskal-Wallis test
Chi-Square test
level of significance: p=0.05

Indices:

See results

Historical control data:

No data

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs.

Mortality:

no mortality observed

Description (incidence):

Scheduled sacrifice of ten out of ten females on gestation day 20.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Significantly reduced body weight gain was noted at >=450 ppm (4 % reduction in final body weight compared to control at 300 ppm, not significant).

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no effects on clinical chemistry parameters.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Relative liver weight was significantly increased at >=300 ppm; there were no effects on absolute liver weight.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathology revealed at >=300 ppm swellings of centrilobular hepatocytes but no degeneration.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no significant effects on implantations or resorption (slight increase at 600 ppm).

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

The number of male live fetuses was significantly decreased in the 600 ppm group, while the number of female live fetuses was not increased in that group.

Dose descriptor:

NOAEC

Effect level:

100 ppm

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEC

Effect level:

300 ppm

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEC

Effect level:

100 ppm

Basis for effect level:

other: developmental toxicity

Dose descriptor:

LOAEC

Effect level:

300 ppm

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

A significant and dose-dependent decrease in fetal body weights of both sexes was not at 300 ppm and above.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

The number of fetuses with anasarca as an external malfomation was significantly increased only in the 600 ppm group.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal malformations were significantly increased in the 450 ppm and 600 ppm group. Adverse effects noted were fused exoccipital and fused cervial arch.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Visceral malformations were significantly increased in the 450 and 600 ppm group. Adverse effects reported were ventricular septal defect, persistent truncus arteriosus, malpositioned subclavian branch, and retroesophageal subclavian. The number of litters having fetuses with cardiovascular malformations was increased in the 450 and 600 ppm groups.

Dose descriptor:

NOAEC

Effect level:

100 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Dose descriptor:

NOAEC

Effect level:

300 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: skull

visceral/soft tissue: cardiovascular

other: external (anasarca); skeletal (cervical arch)

Description (incidence and severity):

Serious cardivascular malformations (ventricular septum defect, persistent truncus arteriosus, malpositioned subclavian branch and retroesophageal subclavian artery), external and skeletal malformations, retarded fetal growth rate and decreased number of male live fetuses.

Developmental effects observed:

yes

Lowest effective dose / conc.:

300 ppm (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Maternal and developmental effects in rats after inhalation exposure to DMAC

Means ± standard deviation (except histopathology)

Parameter	0 ppm	100 ppm	300 ppm	450 ppm	600 ppm
Body weight dams GD 20 (g)	383 ± 21	379 ± 23	368 ± 26	356 ± 16*	330 ± 30*
Abs. liver weight of dams (g)	14.3 ± 1.2	14.6 ± 1.3	15.5 ± 1.5	14.9 ± 1.3	14.5 ± 1.3
Rel. liver weight of dams (g)	3.73 ± 0.20	3.86 ± 0.29	4.22 ± 0.24*	4.19 ± 0.31*	4.43 ± 0.21*
No. of dams with liver swelling	0/10	0/10	4/10	10/10*	7/10*
Live male fetuses/litter	7.4 ± 1.3	7.8 ± 1.3	6.3 ± 2.9	6.9 ± 1.4	4.0 ± 2.8*
Fetal weight (males) (g)	3.90 ± 0.21	3.89 ± 0.20	3.52 ± 0.21*	3.11 ± 0.19*	2.53 ± 0.26*
Fetal weight (females) (g)	3.67 ± 0.16	3.70 ± 0.14	3.36 ± 0.17*	2.89 ± 0.23*	2.46 ± 0.48*

*:significant

Conclusions:

Maternal and developmental effects were found in rats after inhalation exposure at dose levels of 300 ppm (1080 mg/m³), the NOAEC was 100 ppm (360 mg/m³); teratogenic effects at >=450 ppm.

Executive summary:

Developmental toxicity of the test substance was investigated by exposing ten pregnant rats per dose by whole body inhalation to 100, 300, 450, and 600 ppm (v/v) for 6 hours/day during gestation days 6 through 19. Scheduled sacrifice of all animals was performed on gestation day 20. Maternal toxicity was observed with reduced body weight (≥ 450 ppm) and effects on liver (increased relative liver weight and swellings of centrilobular hepatocytes both ≥300 ppm). Neither hepatocellular necrosis nor increased serum activity of liver transaminases was observed in any of the exposed groups. A reduction in fetal body weight and in the number of male live fetuses was a significant observation. The number of fetuses with visceral (cardiovascular), and skeletal (skull, cervial arch) malformations was significantly increased in the 450 and 600 ppm groups, respectively, and the number of fetuses with external (anasarca) was significantly increased in the 600 ppm group. Observed cardiovascular malformations included ventricular septum defect, persistent truncus arteriosus, malpositioned subclavian branch and retroesophageal subclavian artery. The most sensitive signs of developmental toxicity appeared at the exposure level of 300 ppm which was also the level of slight maternal toxicity. The No-Observed-Adverse-Effect-Concentration (NOAEC) was determined as 100 ppm for the endpoints of fetal and maternal toxicities.

Reference 11

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

exposure during gestation day 6-15; no data about sex distribution in offspring

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

- Name of test material: dimethylacetamide (DMAC)
- Source: DuPont
- Purity: 99.9 %
- Impurities: acetic acid, methylamine, water
No further details available.

Species:

rat

Strain:

other: Crl:CD

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at arrival: 60 days old
- Body weight: 165-190 g
- Housing: Individually
- Certified diet and water: ad libitum (not during exposure)
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature: 23-27 °C
- Humidity: 30-50 %
- Air changes: No data
- Photoperiod: 12 hours dark/12 hours light

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION / EXPOSURE
Exposures were conducted in stainless steel chambers, 750 or 900 L; maximum of 55 rats were present in the 750 L chambers, 85 were present in the 900 L chamber; chamber temperature and oxygen concentration were monitored continuously during the exposure; control dams were exposed to air only. Liquid DMAC was syringe-driven through a preheated (200-225°C) inclined tube furnace; nitrogen circulating at 1-2 liters/min carried the vapours through the tube; the DMAC vapour stream was mixed with dilution air at about 500 L/min and then introduced tangentially into the top of the chamber; atmospheres were vented through the bottom of each chamber and passed through an aqueous scrubbing tower to remove DMAC from the exhaust air.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

DMAC concentrations were analyzed using a gas chromatograph (GC) with a flame ionization detector. The GC was calibrated daily by injection of liquid standards; test chambers were analyzed at 30-min intervals by drawing atmospheres through two glass impingers containing acetone and connected in series.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1/1
- Proof of pregnancy: Vaginal plug referred to as day 1 of pregnancy.

Mated females were stratified by body weight and assigned to groups by random sampling within each stratum (25 females per group).

Duration of treatment / exposure:

Gestation days (GD) 6-15

Frequency of treatment:

6 h/day

Duration of test:

GD 21 (termination)

Dose / conc.:

32 ppm (analytical)

Remarks:

Value expressed as mean ± 4 SD. Equivalent to 115 mg/m³. 30 ppm (nominal).

Dose / conc.:

100 ppm (analytical)

Remarks:

Value expressed as mean ± 11 SD. Equivalent to 360 mg/m³. 100 ppm (nominal).

Dose / conc.:

282 ppm (analytical)

Remarks:

Value expressed as mean ± 29 SD. Equivalent to 1015 mg/m³. 300 ppm (nominal).

No. of animals per sex per dose:

25 mated females (24-25 pregnant rats)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A pilot study was conducted with exposure from GD 6-15 at 0, 100, 300 or 625 ppm (parameters measured included maternal signs, external fetal signs, and body weights). At the 625 ppm level, 2 of 4 test females showed complete fetal resorption; dams at 100 or 300 ppm had normal numbers of fetuses.

Maternal examinations:

CLINICAL SIGNS:
Clinical signs were observed daily.

BODY WEIGHT:
Dams were weighed on the day of arrival and on GD 1, 6, 9, 13, 16, and 21.

GROSS PATHOLOGY:
Each dam was coded at termination (blind basis). Dams were euthanized by cervical dislocation. A necropsy was performed.

ORGAN WEIGHTS
Liver was weighed.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination.
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

Ovaries were removed and the number of corpora lutea counted; uterus was opened and the number and position of all live, dead and resorbed conceptuses recorded; gravid and empty uteri were weighed; uterus of each apparently non-pregnant dam was studied for very early resorptions.

Fetal examinations:

All fetuses were weighed.

All fetuses were examined for malformations/variations.
- External examinations: All fetuses
- Soft tissue examinations: About half of the fetuses/litter (plus all stunted and malformed fetuses) (Staples 1974).
- Skeletal examinations: See below.
- Head examinations: About half of the fetuses/litter (fixed in Bouin's solution (Barrow and Taylor 1969)).

All fetuses were sexed internally and then, except for the heads fixed in Bouin's, they were fixed in 70 % ethanol eviscerated, macerated in 1% aqueous KOH, and stained with alizarin red S to permit examination of the skeletons for alterations.
.

Statistics:

The litter used as the experimental unit;
Fisher's exact test
Cochran-Armitage test for trend
Dunnett's test when one-way AN0VA significant
linear trend as tested using the orthogonal polynomial of dose ranks
Mann-Whitney U test
Linear trend was tested using Jonckheere's test
The level of statistical significance was p < 0.05.

Indices:

See results

Historical control data:

No data

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain in dams of the 282 ppm group was significantly reduced.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Liver weight was not altered.

Gross pathological findings:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no treatment related effects on the number of corpora lutea/dam.

Dose descriptor:

NOAEC

Effect level:

100 ppm

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEC

Effect level:

282 ppm

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEC

Effect level:

100 ppm

Basis for effect level:

other: developmental toxicity

Dose descriptor:

LOAEC

Effect level:

282 ppm

Basis for effect level:

other: developmental toxicity

Abnormalities:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): A significant decrease in fetal weight was noted at 282 ppm.

Reduction in number of live offspring:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Dose descriptor:

NOAEC

Effect level:

282 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

no effects observed

Developmental effects observed:

no

Effects on maternal and fetal body weight in rats after inhalation exposure to DMAC

means ± standard error

Parameter tested	0 ppm	32 ppm	100 ppm	282 ppm
Maternal body weight gain (g) GD 6-16	55.7 ± 1.36	53.4 ± 1.50	51.9  ± 2.21	47.3  ± 2.01 *
Fetal body weight (g)	3.54 ± 0.05	3.49 ± 0.06	3.50 ± 0.06	3.30  ± 0.05 *

*: p<0.05

Conclusions:

Maternal and fetotoxic effects but no teratogenicity were noted in rats after inhalation exposure to 282 ppm (1015 mg/m³), the NOAEL was 100 ppm (360 mg/m³).

Executive summary:

The test substance has been reported to be teratogenic when rats were treated via injection or dermal application. The current study investigated groups of pregnant Crl:CD rats exposed to 32, 100, and 282 ppm of the test substance by inhalation for 6 hours/day from Day 6 through Day 15 of gestation. The day on which a copulation plug was observed was termed as Day 1G. A control group of chambered pregnant rats were exposed simultaneously to air only. All maternal rats were sacrificed on Day 21G. Maternal body weight gain and fetal weight were significantly decreased when treated with 282 ppm. The dose-response trend was significant. No effects were observed in animals treated with 32 or 100 ppm. Fetal resorptions were not increased in any of the groups. No treatmetn-related effects were seen in fetuses regarding external, skeletal, and visceral malformations or variations. Therefore, both fetal and maternal toxicity were noted at 282 ppm and the no-observed adverse-effect level (NOAEL) was 100 ppm for both maternal animals and offspring. The test substance did not produce malformations in the rat fetus even at toxic levels to maternal animals.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

65 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

No single study was selected as the weight of evidence approach was applied.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

360 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

No single study was selected as the weight of evidence approach was applied.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Quality of whole database:

The available dermal studies investigating reproductive effects on parental animals was conducted by Industrial Bio-Test Laboratories which was confirmed of engaging in extensive scientific misconduct, thus, the studies were disregarded for assessment.

Additional information

In the OECD SIDS document (2001; SIAM 13) on N,N-dimethylacetamide (DMAC) data were presented on this endpoint which were also documented in this report but more details were presented and an evaluation of the validity of these reports. The evaluation of results is comparable to the OECD SIDS document. Further relevant studies were added.

A bright set of studies is available for the endpoint developmental toxicity/teratogenicity for DMAC. In this endpoint summary only relevant studies are mentioned.

 

Oral exposure route

In a gavage study (1997; RL1: guideline study) 24-25 pregnant Crl:CD®(SD)BR rats per dose level received 0, 20, 65, 150, or 400 mg/kg bw/day at gestation days (GD) 7-21. The study was terminated at GD 22. In dams there were no treatment related clinical signs and no mortality. The body weight gain was significantly and dose dependently reduced at >=150 mg/kg bw/day. Food consumption was significantly reduced at 400 mg/kg bw/day. Clinical chemistry data revealed no treatment related effects. Organ weights showed statistically significant increases in mean absolute kidney weight and in mean relative liver and kidney weights in the 400 mg/kg bw/day dose group. These changes were considered to be test substance-related but not biologically adverse since absolute weight increase was minimal, no histopathological effects were detected, and clinical chemistry data were normal (adaptive response during exposure to DMAC). At necropsy no treatment related effects were detected but placentas from 14 of 25 dams had white or tan outer edges at 400 mg/kg bw/day (consistent with maternal and developmental toxicity). Histopathology of liver and kidney showed increased mitotic figures in livers of dams at 400 mg/kg bw/day which were considered not to be adverse since no degeneration or inflammation was found and clinical chemistry parameters were normal (associated with metabolism of DMAC).

At 400 mg/kg bw/day early and late resorptions were significantly increased resulting in a significantly reduced number of live fetuses; the incidence of fetal malformations was significantly increased as well as the incidence of fetal variations. At >=150 mg/kg bw/day significantly reduced fetal weight was found. Concerning malformations at 150 mg/kg bw/day four affected fetuses from one litter revealed distended brain ventricles (toxicological relevance unclear) and one fetus was found with naris atresia, heart and vessel malformations, cleft palate, macroglossia, micrognathia, and synotia (similarity of specific malformations at the high dose level; authors comment: may represent the bottom end of the dose response curve for malformations).

Conclusion: In a gavage study in rats maternal and developmental toxicity was seen at a dose level of 150 mg/kg bw/day, the NOAEL was 65 mg/kg bw/day.

 

The test subtance was investigated for its teratogenic potential using mice received ten times an oral appliaction of either 256, 427, or 1280 µl/kg bw/day (1976, RL2). Test concentrations were chosen based on an acute toxicity test that has been conducted prior to this study resulting in a LD50 of about 6400 µl/kg bw. Chosen test concentrations were equivalent to 1/5, 1/15 and 1/25 of the LD50. Control animals were included and received water. Oral application of the test substance diluted in water was performed with a cannula and an application volume of 200 µl/animal/day was administered from day 6 until day 15 post coitum leading to a total application frequency of ten times. The operative delivery was performed on day 18 post coitum. All fetuses were observed for external malformations. Two out of three fetuses of one litter each and 1/3 fetus of one litter were observed for skeletal and visceral malformations, variations, and retardation, respectively. The dose of 1280 µl/kg bw/day resulted in a toxic reaction to maternal mice. Body weight gain was reduced and two animals had diarrhea. In fetuses, 1280 µl/kg bw/day led to embryo lethality. Embryos and fetuses died during the entire gestation period. Implantation loss were reported to be 41%. Fetuses delivered on day 18 post coitum showed 77% malformations. A reduced body weight and an increased number of skeletal retardations and variatons were regarded as a sign of fetal toxicity. Maternal animals did not show any signs of toxicity treated with either 427 or 256 µl/kg bw/day. No significant increase in the number of dead fetuses was reported. The number of skeletal and visceral retardations and variations of the fetuses were not correspondent to the dose-independent, however, statistically significant increase of fetal body weight. Fetuses of the 427 µl/lg bw/day group showed weak signs of teratogenicity. Approximately 6% of live fetuses had malformations. Malformations reported for fetuses in the 256 µl/kg bw/day group were similar to the corresponding control group. No maternal toxicity was detected at 400 mg/kg bw/day which was set as NOAEL. Teratogenic effects were detected at 400 mg/kg bw/day and above resulting in a NOAEL of 240 mg/kg bw/day.

 

The test substance was investigated for its prenatal toxicity in rabbits after oral application of thirteen times (1976, RL2). Investigation have been conducted in October 1975 and from January until March 1976 according to guidelines set forth by the FDA (1966). Animals were exposed to 100, 300, and 500 µl/kg bw. The test substance was diluted in bidistilled water and applied via cannula to 10 ml/day from Day 6 to Day 18 post insemination. All animals were sacrified on Day 28 post insemination. All fetuses were observed for external malformations and received two-plane x-ray. Skelteal evaluations were based on x-ray results. The heads of the fetuses were fixed in Bouin'scher solution. Using the method proposed by Wilson (1965), approximately 3 -4 weeks after fixation, ca. 8 -10 transverse cross sections were made and evaluated from each head.

100 µl/kg bw of the test substance were well tolerated by animals without any signs of toxicity. Reduced body weight gain correlated with a reduced food consumption. There were no unscheduled deaths. The number of corpora lutea, implantations, and living fetuses were comparable to control animals. No adverse effects were noted when animals were examined macroscopically. Parameters observed in fetuses (body weight, lenght, weight of placenta, number and types of variations and retardations) were similar to those observed in control animlas. No malformations were reported in fetuses.

300 µl/kg bw of the test substance were tolerated by animals without any signs of toxicity. Reduced body weight gain correlated with a reduced food consumption in a dose-dependant manner. The number of corpora lutea, implantations, and living fetuses were comparable to control animals. The number of living fetuses was reduced and the number of resorptions was increased. Based on recommended number of litters (FDA guideline), the difference was not significant. No adverse effects to the inner organs were reported when animals were examined macroscopically. The average body weight of fetuses was reduced, however, lenght, weight of placenta, and number and types of variations and retardations were normal. No malformations were reported in fetuses.

Animals treated with 500 µl/kg bw of the test substance experienced pain, tremor, and reduced feces as a consequence of an adverse reduction in food consumption. Body weight gain was reduced. One animal died after the eighth and thirteenth application. As a result of early dead fetuses or rather both of the maternal animals, counting of copora lutea was inaccurate, hence, an evaluation was not performed. Conception rate and number of implantations were comparable to control animals. All emryos were resorbed. A defined lobule was investigated in one maternal animals after section. All other examined animals did not show any macroscopic alterations.

Under the conditions chosen, the NOAEL for developmental effects in maternal animals and in the fetus was concluded to be 94 mg/kg bw/day.

 

In supplemental studies developmental toxicity was found in rats at oral dose levels without maternal toxicity; the LOAEL was 320 mg/kg bw/day and the NOAEL for developmental toxicity was 106 mg/kg bw/day. For maternal toxicity the NOAEL was 960 mg/kg bw/day (1976; RL2). Johannsen et al. (1987; RL2) reported developmental and maternal toxicity in rats after gavage of 400 mg/kg bw/day (NOAEL: 160 mg/kg bw/day). In rabbits no maternal toxicity was found after oral exposure via gavage at 280 mg/kg bw/day (NOAEL) but lethal effects at 850 mg/kg bw/day; the NOAEL for developmental effects was 94 mg/kg bw/day, the LOAEL was 280 mg/kg bw/day (1974; RL2). In a special study using single gavage for detection of the most sensitive time during gestation, mice were most susceptible for developmental effects at gestation day 9, the NOAEL for maternal and developmental toxicity was 400 mg/kg bw after single application (1975; RL2).

 

Dermal exposure route

Three studies investigating the developmental toxicity potential of the test substance are available (1972, 1973; RL4). However, the studies were conducted by Industrial Bio-Test Laboratories which was confirmed of engaging in extensive scientific misconduct. Therefore, the studies were not used for further assessment and disregarded due to unreliability of the testing laboratory.

 

Inhalation exposure route

Pregnant Crl:CD rats (24-25 dams per dose level) were exposed for 6 h per day via the inhalation route to 0, 32, 100, or 282 ppm (analytical means corresponding to 0, 115, 360, 1015 mg/m³) at gestation days (GD) 6-15 (Solomon et al., 1991; RL2: comparable to Guideline study but exposure limited to GD 6-15). The study was terminated at GD 21. In dams no clinical signs and no mortality was seen in any test group. Body weight gain in dams of the 282 ppm group was significantly reduced. No effects at necropsy were detected and liver weights were not altered. No treatment related effects were found on number of corpora lutea/dam, implants/litter, resorptions/litter, or live fetuses/litter. A significant decrease in fetal weight at 282 ppm was recorded but no increase in the incidence of malformations or variations in any test group.

Conclusions: There were maternal and fetotoxic effects but there was no teratogenicity in rats after inhalation exposure to 282 ppm (1015 mg/m³), the NOAEC was 100 ppm (360 mg/m³).

 

Ten pregnant Crj:CD(SD)IGS rats per dose level were exposed 6 h per day at gestation days (GD) 6-19 to 0, 100, 300, 450, or 600 ppm (0, 360, 1080, 1620, 2160 mg/m³). The study was terminated at GD 20 (Okuda et al., 2006; RL2: comparable to Guideline study with acceptable restrictions: only 10 dams per dose level; no data about vapour generation; some parameters of developmental toxicity not recorded). No clinical signs were recorded but significantly reduced maternal body weight gain was observed at >=450 ppm. No effects on clinical chemistry parameters were detected. The relative liver weight was significantly increased at >=300 ppm, but no effects on absolute liver weight were seen; histopathology revealed at >=300 ppm swellings of centrilobular hepatocytes but no degeneration. No significant effects on implantations or resorption were reported (slight increase at 600 ppm) but a significant decrease in the number of live fetuses at 600 ppm as well as a significant decrease in fetal weight (males and females) at >=300 ppm. Further examinations of fetuses revealed significant increases in visceral (ventricular septal defect) and skeletal (fused cervical arche) malformations at >=450 ppm; a significant increase in anasarca was found at 600 ppm (external malformation).

Conclusion: Maternal and developmental effects were found in rats after inhalation exposure at dose levels of 300 ppm (1080 mg/m³), the NOAEC was 100 ppm (360 mg/m³); there were teratogenic effects at >=450 ppm (1620 mg/m³).

 

Groups of 15 pregnant rabbits were exposed 6 h per day at gestation days (GD) 7-19 to vapour concentrations of 0, 200, 700, or 2000 mg/m³ (1989; RL2: comparable to Guideline study with acceptable restrictions: 13-14 pregnant rabbits per group; exposure restricted to GD 7-19). The study was terminated at GD 29. Satellite groups (5 rabbits) were exposed to 0 or 2000 mg/m³ and blood samples collected at GD 20 and analysed for clinical chemistry. Histopathology of the liver was performed at termination. No maternal toxicity was found at any dose level and no treatment related developmental effects at <=700 mg/m³.

Fetotoxic effects were caused at a concentration of 0.7 mg/L (increased skeletal variations, e.g. fused sternebrae and accessory ribs), but there was no statistically significant increase in total skeletal variations. At 2.0 mg/L fetotoxic effects (e.g., significantly decreased fetal and placental weights, increase in soft tissue and skeletal variations) and also signs of a weak teratogenic effect expressed as a marginal, statistically not significant increase in soft tissue malformations (regarding the heart and great vessels) were observed. No compound-related effects were observed in the fetuses after exposure to 0.2 mg/l.
Thus, the highest concentration tested under these conditions (2.0 mg/1) was found to be a no-observable-adverse-effectlevel (NOAEL) for the maternal Himalayan rabbit, whereas 0.2 mg/L was defined as the NOAEL for the developing organism.

Overview of the most relevant studies which should be reflected for the DNEL derivation:

Study description / Reference	Result
Prenatal developmental toxicity in rats after oral gavage application from GD 7-21 (termination on GD 22).   OECD test GL 414, GLP study   0, 20, 65, 150, 400 mg/kg bw/day (25 mated rats/group)   DuPont, 1997	NOAEL 65 mg/kg bw/d (maternal, offspring, teratogenicity)
Prenatal developmental toxicity in rats after inhalation exposure from GD 6-19 (termination on GD 20).   OECD test GL 414, GLP study   0, 360, 1080, 1620, 2160 mg/m³ (10 dams/group)   Okuda et al., 2006	NOAEC 360 mg/m³ (maternal, offspring)   Contribution of dermal route due to whole body exposure
Prenatal developmental toxicity in rabbits after inhalation exposure from GD 7-19 (termination on GD 29).   OECD test GL 414, GLP study   0, 200, 700, 2000 mg/m³ (15 mated rabbits/group)   Klimish & Hellwig, 2000 (BG Chemie, 1989)	NOAEL 200 mg/m³ (developmental toxicity) 2000 mg/m³ (maternal)   Contribution of dermal route due to whole body exposure
Prenatal developmental toxicity in rabbits after oral application from GD 6-18 (termination on GD 28).   According to OECD 414, non-GLP   0, 94, 280, 470 mg/kg bw/day, (10 - 11 does per dose group)   BASF, 1976	NOAEL 94 mg/kg bw/day (offspring, teratogenicity) 94 mg/kg bw/day (maternal)
Prenatal developmental toxicity in mice after oral application from GD 6-15 (termination on GD 18).   Equivalent or similar to OECD 414, pre-GLP   0, 240, 400, 1200 mg/kg bw/day, (22 -24 females per dose group)   BASF, 1976	NOAEL 240 mg/kg bw/day (offspring, teratogenicity) 400 mg/kg bw/day (maternal)

Mode of Action Analysis / Human Relevance Framework

no further information

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is considered to reveal no adverse effects on fertility, but it has been proven in animal studies using mice, rabbits, and rats to be teratogenic. Therefore, the test substance is considered to be classified as a reproductive toxicant category 1 B with the hazard statement H360D. The test substance induced external, skeletal, and visceral malformations to experimental animal offspring. It is assumed that all routes of exposure cause the hazard although only reliable data for inhalation and oral exposure are available.

DMAC, CAS 127-19-5 is listed in the Annex VI of Regulation (EC) No. 1272/2008 (CLP Regulation) with the classification for acute dermal and inhalation toxicity Cat. 4*; H312 and H332, and for developmental toxicity / teratogenicity Repr. 1B;H360D.