Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on developmental toxicity

Description of key information

BASF AG (2009): the study with rats and DMA hydrochloride, test concentrations: 100, 300 and 1000 mg/kg bw. A NOAEL for maternal toxicity was 300 mg/kg bw and a NOAEL for developmental toxicity, including teratogenicity was 1000 mg/kg bw.

WIL Research (2016): There were no test substance-related effects on intrauterine growth, survival, and fetal morphology at any exposure concentration; therefore, the NOAEC for embryo/fetal development was 250 ppm for rabbits.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Feb 2008 - 17 Mar 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: Corrigendum to ECC Directive 2004/73/EC, Part B
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
yes
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Time-mated Wistar rats (Crl:WI[Han]) were supplied by Charles River Laboratories, Research Models and Services, Germany GmbH at an age of about 10-15 weeks. Only animals free from clinical signs of disease were used for the investigations.
The animals were paired by the breeder and supplied on GD 0 (= detection of vaginal plug/sperm). After arrival, they were randomly allocated to the test groups by withdrawal from the transport box at random and placed in to a random distribution of groups. After randomization the rats were identified uniquely by ear tattoo.
Reason for species selection: The Crl:WI(Han) strain was selected since extensive experience is available on Wistar rats. This specific strain has been proven to be sensitive to substances with a teratogenic potential.

Acclimatization period: from arrival to GD 6. (so from Day GD 0 to GD 6)

Housing: singly from GD 0-20 in type M III Makrolon cages supplied by BECKER & CO., Castrop-Rauxel, Germany (floor area about 800 cm²).
Bedding: Lignocel FS 14 fibres, dustfree bedding, supplied by SSNIFF, Soest, Germany
Enrichment: wooden gnawing blocks (Typ NGM E-022, supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria).

Accomodation: in fully air-conditioned rooms (central air conditioning)
Temperature: 20-24°C
relative humidity: 30-70%.
Air change rate: 10 times per hour.
The light cycle rhythm was 12 hours light from 6:00 a.m. to 6:00 p.m. and 12 hours darkness from 6:00 p.m. to 6:00 a.m.

Before the study started, the animal room was completely disinfected using a disinfector ("AUTEX" fully automatic, formalin-ammonia-based terminal disinfection). In general, each week the walls and the floor were cleaned with water containing about 0.5% Mikro-Quat (supplied by Ecolab Deutschland GmbH, Hanau, Germany).
Food: ground Kliba maintenance diet mouse/rat “GLP” supplied by PROVIMI KLIBA SA (Kaiseraugst, Switzerland).
Food: available ad libitum throughout the study (from the day of supply to the day of necropsy),
Drinking water: available from water bottles ad libitum

The food used in the study was assayed for chemical and for microbiological contaminants.
The drinking water was regularly assayed for chemical contaminants.
Bedding and the enrichment were regularly assayed for contaminants (chlorinated hydrocarbons and heavy metals).

Based on the pregnant animals the body weight on day 0 varied between 142.5-191.3 g.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
A standard dose volume of 10 mL/kg body weight was used for each group.
The calculation of the volume administered was based on the most recent individual body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Tested by various analyses:
• the stability of the test substance solutions was demonstrated over a period of 10 days at room temperature
• The results of the analyses of the test substance solutions in drinking water confirmed the correctness of the prepared concentrations. Generally, the analytical values of the samples corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations (see PART III; Supplement); except two deviant values in test groups 1 and 2. One deviant value in the samples from the study beginning (test group 2 = 88.8%) and one deviant value in the samples from the end of the study (test group 1 = 80.5%) were considered as outliers with respect to precision and accuracy of the analytical method. As no effects of toxicological concern were observed even at 1000 mg/kg bw/d, these minor deviations had virtually no effect on the quality of the study
Duration of treatment / exposure:
administration from gestational day (GD) 6 through GD 19 = from implantation to one day prior to the expected day of parturition
Frequency of treatment:
once daily
Duration of test:
terminal sacrifice on GD 20
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
nominal in water
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
nominal in water
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
nominal in water
No. of animals per sex per dose:
25 time-mated female Wistar rats per group
Control animals:
yes, concurrent vehicle
Maternal examinations:
Food consumption and body weights of the animals checked regularly (GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20).
Mortality: a check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).
Clinical symptoms: a cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-20).
gross pathology (including weight determinations of the unopened uterus and the placentae).

Sacrifice of the animals on GD 20, then necropsied and assessed by gross pathology.
The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus*
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• live fetuses
• dead implantations:
a) early resorptions (only decidual or placental tissues visible or according to SALEWSKI (Salewski, 1964) from uteri from apparently non pregnant animals and the empty uterus horn in the case of single horn pregnancy)
b) late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) dead fetuses (hypoxaemic fetuses which did not breathe spontaneously after the uterus had been opened)
Ovaries and uterine content:
For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated by resorptions, live and dead fetuses) were determined
Fetal examinations:
Examinations of the fetuses after dissection from the uterus At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus and the base of the genital tubercle and was later confirmed by internal examination, in all fetuses designated for soft tissue examination. If there were discrepancies between the "external" and the "internal" sex of a fetus, the fetus was finally sexed according to the internal sex.

Furthermore, the viability of the fetuses and the condition of placentae, umbilical cords, fetal membranes, and fluids were examined. Individual placental weights were recorded. Thereafter, the fetuses were sacrificed by subcutaneous injection of a pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and placed in ethanol, the other half was placed in Harrison’s fluid for fixation.

Soft tissue examination of the fetuses
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR (Barrow and Taylor, 1969). After this examination these fetuses were discarded.

Skeletal examination of the fetuses
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL (Kimmel and Trammell, 1981). Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.

Classifications based on terms and definitions proposed by CHAHOUD et al. and SOLECKI et al. (Chahoud et al., 1999; Solecki et al., 2001; Solecki et al., 2003):
- Malformation = A permanent structural change that is likely to adversely affect the survival or health.
- Variation = A change that occurs also in fetuses of control animals and is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that has otherwise followed a normal pattern of development.

Moreover, the term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.
All fetal findings were listed in tables according to these classifications.
Statistics:
The conception rate (in %) was calculated according to the following formula:
(number of pregnant animals x 100)/number of fertilized animals
The preimplantation loss (in %) was calculated according to the following formula:
((number of corpora lutea – number of implantations) x 100) / number of corpora lutea
The postimplantation loss (in %) was calculated according to the following formula:
((number of implantations – number of live fetuses) x 100)) / number of implantations

DUNNETT-test (twosided)
FISHER'S EXACT test (one-sided)
WILCOXON-test (onesided)
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: salivation, reduced food consumption, yellowish discolored urine,

Details on maternal toxic effects:
There were no test substance-related mortalities in any of the female animals in any of the groups.
The mean food consumption of the high-dose dams (1000 mg/kg bw/d) was statistically significantly reduced between GD 6 to 8 (14% below control) and GD 8 to 10 (12% below control; see Fig. 4.2.1.3.1.). However, on the following days the food consumption of the high-dose rats became comparable to control. The average food consumption of the highdose dams during the treatment phase (GD 6-19) was less than 4% below the control value. The impaired food consumption is considered to be related to treatment. The food consumption of the females of test groups 1 and 2 (100 and 300 mg/kg bw/d) was unaffected and did not show any statistically significant or biologically relevant differences in comparison to the controls.
Mean body weight and mean body weight gain of low-, mid- and high-dose animals (100; 300 and 1000 mg/kg bw/d) were similar to those of the concurrent controls. All differences observed in these groups during the pretreatment and the treatment period were without biological relevance and reflected the normal variation inherent in the strain of rats used in the present experiment.
The mean gravid uterus weight of the dams of test groups 1; 2 and 3 (100; 300 and 1000 mg/kg bw/d) were comparable to the control and not affected by treatment.
At necropsy, no test substance-related findings were observed in the dams of test groups 0 - 3 (0; 100; 300 and 1000 mg/kg bw/d). One animal of test group 2 showed a diaphragmatic hernia (No. 56). This observation was not considered to be associated to the test compound

The conception rate reached 96% in the control group as well as in test group 1 (100 mg/kg bw/d), 92% in test group 2 (300 mg/kg bw/d) and 100% in test group 3 (1000 mg/kg bw/d). As all presumed pregnant rats had implantation sites at necropsy, a sufficient number of dams were available for the purpose of the study.
No test substance-related and/or biologically relevant differences with regard to conception rate, mean number of corpora lutea, implantation sites, pre- and postimplantation loss and resorptions (total, early and late) were observed

Test group 3 (1000 mg/kg bw/d)
Dams
• Salivation after treatment in 25 dams
• Statistically significant impairment of food consumption between GD 6-8 and GD 8-10.

All animals of the low-, mid- and high-dose groups showed yellowish discoloured urine what has been considered to be treatment-related. Discoloured urine occurred from GD 8 onwards and persisted until the end of the study. This urine discolouration was a sign of systemic availability of the test substance rather than being an adverse effect. It happened most likely due to the excreted test compound or its metabolite(s).
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Remarks on result:
other: see Remarks
Remarks:
based on decreased food consumption and salivation after treatment
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Remarks on result:
other: see Remarks
Remarks:
because no evidence of an adverse effect of the test compound on fetal morphology
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: up to 1000 mg/kg bw /day

Details on embryotoxic / teratogenic effects:
The sex distribution of the fetuses in test groups 1-3 (100; 300 and 1000 mg/kg bw/d) was comparable to the control fetuses. Observable differences were without biological relevance.
The mean placental weights of dose group 1 and 2 (100 and 300 mg/kg bw/d) were similar to the corresponding control. The mean placental weight of the male fetuses of dose group 3 (1000 mg/kg bw/d) was statistically significantly reduced (about 19% below the concurrent control value) but clearly in the range of the historical control data (PART III, Supplement). Therefore, the observed differences were not considered to be biologically relevant and without relation to dosing.
The mean fetal weights were not influenced by the test substance administration and did not show any biologically relevant differences between the test substance-treated groups and the control. The observable differences between the groups reflect the usual fluctuation for this parameter.
External malformations were recorded for one fetus in the control, for four fetuses in the lowdose group (100 mg/kg bw/d) and for four fetuses in the mid-dose group; (300 mg/kg bw/d; Tab. 4.3.2.1.1). Since two low-dose (100 mg/kg bw/d) fetuses of the same litter (dam no. 35) showed gastroschisis and two mid-dose (300 mg/kg bw/d) fetuses of the same litter (dam No. 58) had a menigocele it is reasonable to consider a spontaneous background in single animals rather than a test substance-induced effect. Furthermore, these findings did not show a relation to dosing (Tab. 4.3.2.1.2.). All other findings were incidental or can be found in the historical control data (PART III, Supplement).
No external variations were observed.
No unclassified external observations were recorded.

Soft tissue malformations were recorded for three fetuses in the low-dose group (100 mg/kg bw/d) and for three fetuses in the mid-dose group; (300 mg/kg bw/d; Tab. 4.3.3.1.1.). Since the low- (100 mg/kg bw/d) and the mid-dose (300 mg/kg bw/d) fetuses belonged to the same litters (dam no. 35 and dam no. 58, respectively) and with regard to the findings of the external observation, it is reasonable to consider a spontaneous background in single animals rather than a test substance-induced effect due to a missing dose-response relationship (Tab. 4.3.3.1.2.).

Fetuses
• No test substance-related adverse effects Test group 2 (300 mg/kg bw/d)
• No test substance-related adverse effects on dams, gestational parameters or fetuses Test group 1 (100 mg/kg bw/d)
• No test substance-related adverse effects on dams, gestational parameters or fetuses
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Embryotoxic / teratogenic effects:no effects
Abnormalities:
not specified
Developmental effects observed:
not specified

Malformations of the fetuses: cleft palate, gastroschisis, exencephaly, mandibular micrognathia, malrotated limb (bilateral hindlimb) meningocele.

Soft tissue malformations:

Fetus with multiple visceral malformations: short intestine, large uterus horns (reaching to the middle of the kidneys), enlarged ovaries, persistent truncus arteriosus, heart: muscular ventricular septum defect, anophthalmia (bilateral),

Fetus with multiple visceral malformations: absent subclavian, persistent truncus arteriosus, heart: muscular ventricular septum defect, anophthalmia (bilateral),

anophthalmia (left)

Soft tissue variation:

Four soft tissue variations were detected, i.e. dilated cerebral ventricle, short innominate and uni- or bilateral dilation of renal pelvis and ureter. Dilated cerebral ventricle and short innominate occurred only in the male fetus 35-07 of the low-dose group and the male fetus 58-05 of the mid-dose group in addition to external, soft tissue and skeletal malformations. Uni- or bilateral dilation of renal pelvis and ureter were seen in several fetuses of all test groups including the control. These findings were considered to be incidental because they did not show a relation to dosing. (Tab. 4.3.3.2.1.). In addition, they can be found in the historical control data (PART III, Supplement) in comparable or even higher incidences.

Fetal skeletal malformations:

Skeletal malformations were noted in fetuses of test groups 1 and 2 (100 and 300 mg/kg bw/d; Tab. 4.3.4.1.1.). No dose-response relationship was observed (Tab. 4.3.4.1.2.). Based on the rate of affected fetuses per litter, the incidence of skeletal malformations was comparable to the historical control data (PART III, Supplement).

Fetal skeletal variations:

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dosing (Tab. 4.3.4.2.1.). Based on the rate of affected fetuses per litter, the incidence of skeletal variations was comparable to the historical control data (PART III, Supplement).

Fetal skeletal unclassified cartilage observation:

Two isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, were noted in all test groups or only in test group 2. These cartilage findings, i.e. bipartite processus xiphoideus and notched manubrium were related to the sternum. An association to the test substance is not assumed because the incidences of both observations were within the historical control range (PART III, Supplement).

Conclusions:
Thus, the oral administration of Dimethylamine hydrochloride to pregnant Wistar rats had no effect on morphology of offspring at any dose level tested (100; 300 and 1000 mg/kg bw/d). The recorded incidences did not suggest a treatment-relationship, but reflected the usual biological variation inherent in the strain of rats used for this experiment.
In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 300 mg/kg bw/d based on decreased food consumption and salivation after treatment in the highdose dams (1000 mg/kg bw/d). The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 1000 mg/kg bw/d because there was no evidence of an adverse effect of the test compound on fetal morphology.
Executive summary:

Dimethylamine hydrochloride was administered to pregnant Wistar rats daily by gavage from implantation (GD 6) to one day prior to the expected day of parturition (GD 19). The test substance did not cause any mortality. Test substance-related relevant clinical effects were only seen in the high-dose dams (1000 mg/kg bw/d), i.e. salivation after treatment and decreased food consumption, although the latter did not affect body weight, body weight gain, net body weight gain and uterus weight. At necropsy, no test substance related findings were noted in any of the dams. The temporary salivation was likely to be induced by the taste of the test substance or by local irritation of the upper digestive tract. It was not considered to be a sign of systemic toxicity. All animals of the low-, mid- and high-dose groups showed yellowish discoloured urine which occurred from GD 8 onwards and persisted until the end of the study. The urine discolouration was a sign of systemic availability of the test substance and happened most likely due to the excreted test compound or its metabolite(s). This finding has been considered to be treatment-related but was not assessed as an adverse effect. No differences of toxicological relevance between the control and the dose groups were determined for reproductive parameters such as conception rate, mean number of corpora lutea, mean number of implantations, pre- and postimplantation losses, live fetuses and fetal sex ratio. Examination of the fetuses revealed incidental fetal external, soft tissue and skeletal malformations in individual litters of the low- and the mid-dose groups as well as the control. Since malformations only occurred in one litter of the low- and one litter of the mid-dose group, it is reasonable to consider a spontaneous background in single animals rather than a test substance-induced effect. A consistent pattern and a dose-response relationship were missing. Thus, a test substance-related effect on ontogeny is not assumed. No external variation was noted. Four soft tissue and a broad range of skeletal variations occurred in every test group including the control. All of these variations are documented at a comparable frequency in the historical control data (Part III, Supplement). A spontaneous origin is also assumed for the unclassified cartilage observations, which were recorded for fetuses of all dose-groups including the control. Character as well as distribution of all of these findings did not suggest a relation to treatment. In summary, there was no evidence of an adverse effect of Dimethylamine hydrochloride on fetal morphology at any dose level tested.

Thus, the oral administration of Dimethylamine hydrochloride to pregnant Wistar rats had no effect on morphology of offspring at any dose level tested (100; 300 and 1000 mg/kg bw/d). The recorded incidences did not suggest a treatment-relationship, but reflected the usual biological variation inherent in the strain of rats used for this experiment. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 300 mg/kg bw/d based on decreased food consumption and salivation after treatment in the highdose dams (1000 mg/kg bw/d). The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 1000 mg/kg bw/d because there was no evidence of an adverse effect of the test compound on fetal morphology.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Remarks:
US EPA EPA GLP Standards 40 CFR Part 160 and 40 CFR Part 792 (16-Oct-1989 and 18-Sep-1989, respectively) and the OECD Principles of GLP [C(97) 186/Final] (26-Nov-1997)
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: New Zealand White [Hra:(NZW)SPF]
- Source: Covance Research Products, Inc., Greenfield, IN
- Age at study initiation: approximately 6 months old upon receipt.
- Weight at study initiation: Body weight values ranged from 2900 g to 3878 g on gestation day 0.
- Fasting period before study: no, food was withheld only during exposure periods.
- Housing: Upon arrival, all rabbits were housed individually in clean, stainless steel cages suspended above ground corncob bedding (Pel O’Cobs®; The Andersons, Cob Products Division, Maumee, OH). During exposure period, animals were individually housed in stainless steel wire mesh caging.
- Diet (e.g. ad libitum): The basal diet: PMI Nutrition International, LLC Certified Rabbit LabDiet® 5322. The basal diet was offered in 25-g increments 3 times per day on the day of arrival and in increased amounts over the next few days, until the animals gradually achieved ad libitum status prior to the exposure period; basal diet was offered ad libitum throughout the study, except during the exposure periods when food was withheld.

Kale (1 leaf at each occasion) was provided to each animal daily for environmental enrichment and to aid in maintaining the animal's gastrointestinal health, beginning upon animal receipt and continuing throughout the duration of the study. Kale present in the cage was discarded at the time of providing a new leaf.
Use of non-certified kale did not have an adverse impact on the quality or integrity of the data or the outcome of the study as kale is commonly regarded as safe and is intended for human consumption.

- Water (e.g. ad libitum): Municipal water. Reverse osmosis purified (on site) drinking water, delivered by an automatic watering system, was provided ad libitum throughout the study, except during the exposure periods when water was withheld.
- Acclimation period: not specified. However, it is reported that animals were received on gestation day 3 or 4 and the exposure started from gestation day 7.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C ± 3° (66°F ± 5°F)
- Humidity (%): 50% ± 20%,
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
21-Sep-2015 to 13-Oct-2015 (Test substance exposure period (Phase I))
02-Nov-2015 to 24-Nov-2015 (Test substance exposure period (Phase II))
Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: humidified filtered air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposures were conducted using four 1500-L glass and stainless steel whole-body exposure chambers. One 1500-L chamber was dedicated to the filtered-air control group and three 1500-L chambers were dedicated to each of the 3 exposure levels. One chamber was dedicated for each group for the duration of the study. Chamber supply air was provided from a HEPA- and charcoal-filtered, temperature- and humidity-controlled source. All exposure chamber exhaust passed through the facility exhaust system that included charcoal- and HEPA-filtration units.

- Method of holding animals in test chamber: Animals were individually housed in stainless steel wire mesh caging and food and water were withheld during the exposure periods. Animals were housed in a normal animal colony room during non-exposure hours. Prior to each exposure, the animals were transferred to exposure caging and transported to the exposure room. Animals were then exposed for the requisite duration and returned to their home cages in the animal colony room. Animals were housed individually in standard exposure batteries of appropriate size for the whole-body chamber in use during exposure periods. To ensure a similar exposure for all animals, the exposure batteries were rotated daily amongst 3 chamber positions.

- Source and rate of air: Test substance atmosphere was generated by releasing test substance gas (1,000,000 ppm) from the original cylinder. The gas cylinder was heated using heating pads controlled by temperature controllers and J-Type thermocouples. A 2 stage regulator with pressure gauges was used to control test substance flow from the cylinder to a needle valve through 1/8-inch stainless steel tubing. Test substance from the regulator was delivered to a manifold system equipped with a pressure gauge to monitor manifold pressure. The manifold delivery line was heated using a heat tape with a temperature controller and a J Type thermocouple. The manifold was used to distribute test substance to each exposure chamber through 1/8-inch Teflon® tubing. The test substance gas flow to the manifold was controlled using a needle valve and metered using rotameter-type flowmeters.

- Temperature, humidity, pressure in air chamber: Temperature, relative humidity, chamber ventilation rate, and negative pressure within the exposure chambers were continually monitored and recorded approximately every 45 minutes during the 6-hour exposure periods. The mean temperature and mean relative humidity were to be between 16°C to 22°C and 30% to 70%, respectively.

- Air flow rate: Chamber airflow rates were monitored using a sharp edge orifice meter and Dwyer Magnehelic® Indicating Transmitter pressure gauge (Dwyer Instruments, Inc.; Michigan City, IN). Each gauge was calibrated for conversion from pressure to airflow in standard liters per minute through the use of a Fox Gas Mass Flowmeter Transmitter (Model FT2, Fox Thermal Instruments; Marina, CA).

- Air change rate: at least 12 air changes per hour.

- Treatment of exhaust air: Test substance gas was directed to the chamber inlet through a 3-way valve where it was mixed and diluted with facility dilution supply air. The 3-way valve was used to divert the flow of the test substance gas from the chamber directly to facility exhaust, if necessary. The test substance delivery lines from the 3-way bypass valve to the chamber inlet were 1/4 inch stainless-steel tubing heated using a heat tape with temperature controllers and J-Type thermocouples.
- Oxygen content was measured during the method development phase and was 20.9% for all chambers.

TEST ATMOSPHERE
- Brief description of analytical method used: GC
- Samples taken from breathing zone: yes.

VEHICLE (if applicable)
The control substance used for exposure of the control group (Group 1) was humidified, filtered air.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Due to the use of a single cylinder of test substance for generation of all test substance atmospheres, nominal concentrations were not calculated. Test substance usage was documented in the study records.

Analyzed exposure concentrations were determined at approximately 45-minute intervals using an appropriate gas chromatography (GC) method. Samples were collected from the approximate animal breathing zone of each exposure chamber via 1/8-inch Teflon® tubing. Exposure atmosphere samples were collected automatically using an external multi position valve. Gas sample injection into the chromatography column occurred via an internal gas-sampling valve with a sample loop. The chromatograph was displayed, the area under the sample peak was calculated and stored, and the concentration in parts per million (ppm) was calculated.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
once a day
Duration of test:
during gestation days 7-28
Dose / conc.:
0 ppm (analytical)
Remarks:
Due to the use of a single cylinder of test substance for generation of all test substance atmospheres, nominal concentrations were not calculated. Test substance usage was documented in the study records.
Dose / conc.:
50 ppm (analytical)
Remarks:
Due to the use of a single cylinder of test substance for generation of all test substance atmospheres, nominal concentrations were not calculated. Test substance usage was documented in the study records.
Dose / conc.:
100 ppm (analytical)
Remarks:
Due to the use of a single cylinder of test substance for generation of all test substance atmospheres, nominal concentrations were not calculated. Test substance usage was documented in the study records.
Dose / conc.:
250 ppm (analytical)
Remarks:
Due to the use of a single cylinder of test substance for generation of all test substance atmospheres, nominal concentrations were not calculated. Test substance usage was documented in the study records.
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: pre-test:
In WIL 235503 (Weinberg, 2015), nonpregnant rabbits were exposed to 50, 100, 150, 300, and 700 ppm of the test substance atmospheres for 6-hours (whole-body) per day for 10 consecutive days. Exposure of 700 ppm was not tolerated and the animals were euthanized prior to the end of the first day of exposure. On study day 1, observations of labored respiration were noted at the 300 ppm exposure concentration, but all rabbits survived and this observation was not noted following the first day of exposure. There were no gross observations at necropsy from the rabbits that survived to scheduled euthanasia. Based on these data, exposure concentrations of 50, 150, 200, and 250 ppm were selected for a range-finding study in pregnant rabbits (Charlap, 2015, WIL-235501). All animals survived to scheduled euthanasia. Decreased respiration was noted in animals at the approximate mid-point of exposure observation from 150 ppm through 250 ppm (highest exposure concentration). Additional observations at the approximate mid-point of exposure observation included clear nasal discharge in the 250 ppm group and wet clear material around the nose in the 150 ppm group (although only a single incidence) through the 250 ppm group. Clear material around the mouth was also noted in 200 and 250 ppm groups. Observations at 1 2 hours following exposure included rales in the 200 and 250 ppm groups and clear material around the nose in the 150, 200, and 250 ppm groups. Body weight and food consumption data were generally comparable across groups.
Based on these data, target exposure concentrations of 0, 50, 100, and 250 ppm were selected for this definitive study.

- Rationale for animal assignment (if not random): each animal judged to be in good health and meeting acceptable gestation day 0 body weight requirements was selected for use in the computerized randomization procedure based on body weight stratification in a block design.
Separate randomizations were conducted for Phases I and II. Replacement animals were arbitrarily assigned based on body weight prior to the initiation of exposure

- Other: the study was conducted in two phases:
The number of animals selected for this study (24 females/group) was based on the United States EPA Health Effects Test Guidelines: OPPTS 870.3700, Prenatal Development Toxicity Study, Aug-1998 and the OECD Guidelines for the Testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, Jan 2001, which recommend evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 20 at termination. In addition, due to limitation of exposure chamber size, only 12 rabbits could be placed into each exposure chamber; therefore, 24 rabbits was the maximum number of animals per group that could be placed on study to allow completion of the study in 2 phases.
Fifty-two time-mated female New Zealand White rabbits were received in good health from Covance Research Products, Inc., Greenfield, IN, on 18-Sep-2015 (first shipment; Phase I). An additional 52 rabbits of the same strain were received from the same supplier on 30-Oct-2015 (second shipment; Phase II). The time-mated rabbits were received on gestation day 3 or 4; a breeding record was provided by the supplier and is maintained in the study records.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rabbits were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were recorded daily from the day of receipt through gestation day 29 (prior to exposure during the treatment period). Animals were also observed for signs of toxicity at the approximate midpoint of exposure and 1-2 hours following exposure. The absence or presence of findings was recorded for individual animals at observations conducted 1-2 hours following exposure. Only significant findings were recorded at the approximate midpoint of exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 (by supplier under conditions that were not compliant with GLPs, but in accordance with the supplier’s SOPs), 4, and 7-29 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 7-10, 10-13, 13-20, 20-29, and 7-29.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Individual food consumption was recorded on gestation days 4-29.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

POST-MORTEM EXAMINATIONS: Yes
The laparohysterectomies and macroscopic examinations were performed blind to treatment group.
- Sacrifice on gestation day 29
- Organs examined: The thoracic, abdominal, and pelvic cavities were opened by a ventral mid line incision, and the contents were examined.

OTHER: Ocular Irritation Observations
Examination of ocular irritation in both eyes of all animals was performed on gestation day 3 or 4 (day of animal receipt) and gestation day 28 in accordance with the method of Draize (1965) and was facilitated by use of a direct ophthalmoscope, as necessary. Sodium fluorescein was used to aid in revealing possible corneal injury at all examinations.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
The thoracic, abdominal, and pelvic cavities were opened by a ventral mid line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised.
Examinations included:
The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
- Gravid uterus weight: Yes
Gravid uterine weight was collected and net body weight (the gestation day 29 body weight exclusive of the weight of the uterus and contents) and net body weight change (the gestation day 0 29 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy.

- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).
Maternal tissues were preserved in 10% neutral buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.
Fetal examinations:
- External examinations: Yes: [all per litter]: each viable fetus
Fetal examinations were performed blind to exposure group. Each viable fetus was examined externally, individually weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the WIL Research study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown rump measurements, degrees of autolysis and gross examinations, if possible, were recorded for late resorptions, and the tissues were discarded.

- Soft tissue examinations: Yes: [all per litter]: each viable fetus
Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels (Stuckhardt and Poppe, 1984). The sex of each fetus was determined by internal examination. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972).

- Skeletal examinations: Yes: [all per litter]
Following fixation in alcohol, each fetus was stained with Alizarin Red S (Dawson, 1926) and Alcian Blue (Inouye, 1976). Fetuses were then examined for skeletal malformations and developmental variations.

- Head examinations: Yes: [half per litter]
Heads from approximately one half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft tissue examination by the Wilson sectioning technique (Wilson, 1965). The heads from the remaining one half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.
Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance exposed group to the control group. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid animals were excluded from statistical analyses.
Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance exposed groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre and post implantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined), and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance exposed groups to the control group.
Indices:
Intrauterine data were summarized using 2 methods of calculation:
- Postimplantation Loss/Litter based on 1) group mean litter basis and on 2) proportional litter basis (in %);
- Sum of postimplantation losses per group was calculated based on proportional litter basis (please see below table 1).

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as presented in table 2 (please see below).
Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: the higher incidence clinical observations (rales, elevated head, and clear material findings), mean body weight losses, and lower food consumption in the 250 ppm group.

Details on maternal toxic effects:
All females in the control and test substance-exposed groups survived to the scheduled necropsy on gestation day 29.
Test substance-related adverse rales were noted in 250 ppm group at the daily examinations and at 1-2 hours following exposure throughout the exposure period. An increase in the incidence of elevated head and clear material around the mouth was noted at the approximate mid-point of each daily exposure for the 250 ppm group throughout the exposure period compared to the control group. Clear material around the eyes was noted primarily for the 250 ppm group at 1-2 hours following exposure and at the daily examinations beginning on gestation day 14 and continuing through euthanasia. Clear material around the nose was noted for all test substance-exposed groups at 1-2 hours following exposure throughout the exposure period; this finding was also noted for the majority of females in the 250 ppm group at the daily examinations and the mid-point of exposure.
Other clinical findings, including brown, red, and or yellow material on various body surfaces (nose and/or anogenital region), occurred infrequently and/or in a manner that was not exposure-related.

A test substance-related significant (p<0.01) mean body weight loss was noted in the 250 ppm group during the gestation day 7-10 interval compared to a mean body weight gain in the control group. Mean body weight gains in the 250 ppm group were similar to the control group throughout the remainder of the treatment period (gestation days 10-29). The initial mean body weight loss resulted in a significantly (p<0.05) lower mean body weight gain for the entire exposure period (gestation day 7-29) at 250 ppm. However, the mean body weight loss was not of sufficient magnitude to result in a lower mean body weight at the end of the exposure period. In addition, a larger mean net body weight loss (not statistically significant) was noted in the 250 ppm group when compared to the control group. Mean net body weight and gravid uterine weight in this group were similar to the control group.
Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 50 and 100 ppm groups were unaffected by test substance exposure. Differences from the control group were slight and not statistically significant, with the following exception. A significantly (p<0.05) higher mean body weight gain was noted in the 100 ppm group during gestation day 21-22. This difference was transient and not dose-responsive.

Test substance-related lower mean food consumption (g/animal/day and g/kg/day) was noted in the 250 ppm group during the first 5 days of exposure (gestation days 7-12; differences were generally significant [p<0.05 or p<0.01]). For the remainder of the exposure period slightly lower mean food consumption was noted for the 250 ppm group compared to the control group; differences were significant (p<0.05) during gestation days 19-20 and 20-21 (g/animal/day only). Lower mean food consumption noted in the 250 ppm group correlated to lower mean body weight gains during the exposure period.
Food consumption, evaluated as g/animal/day and g/kg/day, in the 50 and 100 ppm groups was unaffected by test substance exposure. Differences from the control group were slight and not statistically significant.

Details on ocular irritation observations, maternal necropsy data and on laparohysterectomy on gestation day 29 are presented below in the "Any other information on results incl.tables".
Dose descriptor:
NOAEC
Effect level:
100 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
250 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: no statistically significant malformations or developmental variations were atributed to the test substance.

Details on embryotoxic / teratogenic effects:
The numbers of fetuses (litters) available for morphological evaluation were 209(21), 191(22), 211(23), and 195(21) in the control, 50, 100, and 250 ppm groups, respectively. Malformations were observed in 7(7), 3(3), 7(4), and 6(3) fetuses (litters) in these same respective exposure groups and were considered spontaneous in origin. When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the control group were noted. Fetal malformations and developmental variations, when observed in the test substance exposed groups, occurred infrequently or at a frequency similar to that in the control group, did not occur in an exposure-related manner, and/or were within the WIL Research historical control data ranges (Appendix F). Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Dose descriptor:
NOAEC
Effect level:
250 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Embryotoxic / teratogenic effects:no effects.
Abnormalities:
not specified
Developmental effects observed:
not specified

The overall mean analyzed concentrations for each group during Phases I and II are presented below:

Table 1. Mean Analyzed Exposure Concentration (Phase I)

Exposure System:

1

2

3

4

Target Concentration (ppm):

0

50

100

250

Study Mean Concentration (ppm):

0

50

103

251

Standard Deviation:

0.0

5.4

4.3

7.9

N:

23

23

23

23

 

Table 2. Mean Analyzed Exposure Concentration (Phase II)

Exposure System:

1

2

3

4

Target Concentration (ppm):

0

50

100

250

Study Mean Concentration (ppm):

0

50

100

247

Standard Deviation:

0.0

3.0

2.7

7.2

N:

23

23

23

23

Maternal data

Ocular Irritation Observations

There were no test substance-related effects on the eyes for any animals. There were some animals that were noted with conjunctival hyperemia/congestion or corneal opacity in the test substance groups. However, the findings were noted in a manner that was not exposure-related, occurred in single animals, and/or occurred at a similar incidence in the control group or during the pre-exposure assessment on gestation day 3 or 4.

 

At the scheduled necropsy on gestation day 29, no test substancerelated internal findings were observed at any exposure level. Macroscopic findings observed in the test substanceexposed groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not exposurerelated. All females were gravid with the exception of 3, 2, 1, and 3 female(s) in the control, 50, 100, and 250 ppm groups, respectively.

 

Gestation Day 29 Laparohysterectomy

Intrauterine growth and survival were unaffected by test substance exposure at exposure levels of 50, 100, and 250 ppm. Parameters evaluated included post implantation loss, live litter size, mean fetal body weights, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites were similar across all groups. Mean litter proportions of pre-implantation loss were higher in the test substance-exposed groups (9.9%, 10.5%, and 7.7% for the 50, 100, and 250 ppm groups, respectively) compared to the control group (2.3%) but were not consider test substance-related because the initiation of exposure started after implantation and the values in the test substance groups were similar to the WIL Research historical control mean (8.1 ± 2.95% per litter; Appendix F). All other differences from the control group were slight and not statistically significant.

 

Fetal morphological data

The numbers of fetuses (litters) available for morphological evaluation were 209(21), 191(22), 211(23), and 195(21) in the control, 50, 100, and 250 ppm groups, respectively. Malformations were observed in 7(7), 3(3), 7(4), and 6(3) fetuses (litters) in these same respective dosage groups and were considered spontaneous in origin.

 

External Malformations and Variations

External malformations were limited to 2 fetuses in the 100 ppm group and 1 fetus in the 250 ppm group. Fetus nos. 3178-02 and 3178-07 in the 100 ppm group were noted with disseminated subcutaneous hemorrhage. In the 250 ppm group, fetus no. 5220-09 was noted with a short tail; skeletally, this finding consisted of absent, fused, small, and misshapen caudal vertebra. These malformations were noted in single fetuses or litters and/or in a manner that was not exposure-related, and therefore were not considered test substancerelated.

There were no external developmental variations noted for fetuses at any exposure level.

 

Visceral Malformations and Variations

Visceral malformations were observed in 7(7), 3(3), 1(1), and 3(2) fetuses (litters) in the control, 50, 100, and 250 ppm groups, respectively. A visceral malformation of diaphragmatic hernia (portion of left lobe of liver and stomach protruded into the thoracic cavity though an opening in the diaphragm) was noted for 2 fetuses(nos. 5220-06 and 5220-09) from the same litter in the 250 ppm group. The incidence of this malformation at 250 ppm(1.2% per litter) was slightly outside of the WIL Research historical data range (0% to 1.0% per litter; Appendix F). However, the incidence was not statistically significant when compared to the concurrent control group, diaphragmatic hernia has been previously observed in 2 fetuses from 1 control group in the WIL Research historical control data (version 4.3 full), and this finding is the second most common visceral malformation observed in the WIL Research historical control data (Appendix F). In addition, no other effects on fetal morphology were observed in the 250 ppm group. Therefore, the 2 diaphragmatic hernias observed in a single litter in the 250 ppm group were not considered to be test substance-related.

Lobular agenesis of the lungs (right accessory lobe absent) was noted for 6(6), 2(2), 1(1), and 1(1) fetuses (litters) in the control, 50, 100, and 250 ppm groups, respectively. An enlarged heart (all chambers), interventricular septal defect (an opening in the anterior portion of the septum), and misshapen papillary muscles were noted for fetus no. 5214-01 in the 50 ppm group. The aforementioned visceral malformations were not consisted test substance-related because they occurred in single fetuses, similarly in the control group, and/or in a manner that was not dose-related. Fetus no. 5232-09 in the control group was noted with folded retina.

No test item-related visceral developmental variations were noted. Findings observed in the test itemtreated groups were noted infrequently, similarly in the control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data (Appendix F).Red fluid abdominal cavity was noted for a single fetus (no. 5214-01) in the 50 ppm group. This finding was not classified as either a malformation or developmental variation, was not included on the summary tables, and was not considered to be test substance-related because it occurred in single fetus and in a manner that was not exposure-related. Skeletal Malformations and Variations Skeletal malformations were noted for 4(2) and 4(3) fetuses (litters) in the 100 and 250 ppm groups, respectively. Fetus no. 3156-10 in the 100 ppm group and fetus nos. 3168-03 and 5220-06 in the 250 ppm group were noted with vertebral anomalies without associated rib anomalies(absent, extra, fused, mal positioned, or mal proportioned arches or fused, extra, misshapen, mal positioned, or mal proportioned centrum). Fetus nos. 3156-04 and 5235-01 in the 100 ppm group and fetus no. 3168-08 in the 250 ppm group were noted with vertebral central anomalies(bipartite, fused, mal positioned, or mal proportioned centrum). Fetus no. 3156-13 in the 100 ppm group was noted with severely maligned sternebra(e). Fetus no. 5194-02 in the 250 ppm group was noted with a rib anomaly (forked rib and mal positioned costal cartilage) and costal cartilage anomaly (right costal cartilage no. 7 bifurcates, rejoins and associates with sternum in normal no. 7 position). The aforementioned malformations were noted in a single fetus and/or mean litter proportions were within the WIL Research historical control data ranges(Appendix F)and therefore they were not considered test substancerelated. No test substance-related skeletal developmental variations were noted. Findings observed in the test substanceexposed groups were noted infrequently, similarly in the control group, were not observed in an exposure-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the WIL Research historical control data (Appendix F).

Conclusions:
Based on the higher incidence clinical observations (rales, elevated head, and clear material findings), mean body weight losses, and lower food consumption in the 250 ppm group, an exposure level of 100 ppm was considered to be the no observed adverse effect concentration (NOAEC) for maternal toxicity. There were no test substance-related effects on intrauterine growth, survival, and fetal morphology at any exposure concentration; therefore, the NOAEC for embryo/fetal development was 250 ppm when dimethylamine was administered via whole body inhalation exposure for 6 hours per day from gestation days 7 through 28 to time-mated New Zealand White rabbits.
Executive summary:

The objectives of the study were to determine the potential of the test substance, dimethylamine, to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect concentration (NOAEC) for maternal toxicity and developmental toxicity (WIL Research, 2016).

Dimethylamine (DMA) was administered via whole-body inhalation exposure to 3 groups of 24 time-mated female New Zealand White [Hra:(NZW)] rabbits for 6 hours per day from gestation days 7 through 28. Target exposure concentrations were 50, 100, and 250 ppm for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) composed of 24 time-mated females was exposed to humidified, filtered air on a comparable regimen. Due to limitation of exposure chamber size, only 12 rabbits could be placed into each exposure chamber; therefore, this study was conducted in 2 phases, with 12 animals/group in each phase. The females were approximately 6 months of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. The eyes of all animals were examined for ocular irritation on gestation day 3or 4 (day of animal receipt) and gestation day 28. On gestation day 29, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

All females in the control and test substance-exposed groups survived to the scheduled necropsy on gestation day 29.

Rales were noted in 250 ppm group at the daily examinations and at 12 hours following exposure throughout the exposure period. An increase in the incidence of elevated head was also noted in the 250 ppm group at the mid-point of exposure throughout the exposure period. Clear material around the eyes, mouth, and nose were noted at the daily examinations, approximate mid-point of each daily exposure, and/or 12 hours following exposure for the 250 ppm group throughout the exposure period compared to the control group. The aforementioned clinical observations at 250 ppm were considered test substance-related and adverse. In addition, test substance-related clear material around the nose was noted for 50 and 100 ppm groups at 12 hours following exposure throughout the exposure period. In the absence of other signs of toxicity at 50 and 100 ppm, and because the material findings did not persist to the daily examinations, they were not considered adverse at these exposure levels.

Test substance-related mean body weight losses were noted in the 250 ppm group during gestation days 7-10 compared to mean body weight gains in the control group. Corresponding lower mean food consumption was noted in this group generally throughout the exposure period. The initial mean body weight losses in the 250 ppm group were not of sufficient magnitude to result in lower mean body weight at the end of the exposure period. A mean net body weight loss was noted in the 250 ppm group. Mean maternal body weights, net body weights, and gravid uterine weights in all test substance-exposed groups and mean body weight gains and net body weight changes in the 50 and 100 ppm groups were unaffected by test substance exposure.

There were no remarkable ocular or macroscopic findings in any exposure group.

Intrauterine growth, survival, and fetal morphology (external, visceral, and skeletal) were unaffected by test substance exposure at all exposure levels.

Based on the higher incidence clinical observations (rales, elevated head, and clear material findings), mean body weight losses, and lower food consumption in the 250 ppm group, an exposure level of 100 ppm was considered to be the noobservedadverseeffect concentration (NOAEC) for maternal toxicity. There were no test substance-related effects on intrauterine growth, survival, and fetal morphology at any exposure concentration; therefore, the NOAEC for embryo/fetal development was 250 ppm when dimethylamine was administered via wholebody inhalation exposure for 6 hours per day from gestation days 7 through 28 to time-mated New Zealand White rabbits.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The overal quality of the database is high because two GLP guideline studies available: in one species (rat) and in second species (rabbit).
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
461 mg/m³
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The overal quality of the database is high because two GLP guideline studies available: in one species (rat) and in second species (rabbit).
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Additional information

Oral developmental toxicity study in one species (rats)

In a GLP OECD 414 study, Dimethylamine hydrochloride was administered to pregnant Wistar rats daily by gavage from implantation (GD 6) to one day prior to the expected day of parturition (GD 19) (BASF AG, 2009). The test substance did not cause any mortality. Test substance-related relevant clinical effects were only seen in the high-dose dams (1000 mg/kg bw/d), i.e. salivation after treatment and decreased food consumption, although the latter did not affect body weight, body weight gain, net body weight gain and uterus weight. At necropsy, no test substance related findings were noted in any of the dams. The temporary salivation was likely to be induced by the taste of the test substance or by local irritation of the upper digestive tract. It was not considered to be a sign of systemic toxicity. All animals of the low-, mid- and high-dose groups showed yellowish discoloured urine which occurred from GD 8 onwards and persisted until the end of the study. The urine discoloration was a sign of systemic availability of the test substance and happened most likely due to the excreted test compound or its metabolite(s). This finding has been considered to be treatment-related but was not assessed as an adverse effect. No differences of toxicological relevance between the control and the dose groups were determined for reproductive parameters such as conception rate, mean number of corpora lutea, mean number of implantations, pre- and postimplantation losses, live fetuses and fetal sex ratio. Examination of the fetuses revealed incidental fetal external, soft tissue and skeletal malformations in individual litters of the low- and the mid-dose groups as well as the control. Since malformations only occurred in one litter of the low- and one litter of the mid-dose group, it is reasonable to consider a spontaneous background in single animals rather than a test substance-induced effect. A consistent pattern and a dose-response relationship were missing. Thus, a test substance-related effect on ontogeny is not assumed. No external variation was noted. Four soft tissue and a broad range of skeletal variations occurred in every test group including the control. All of these variations are documented at a comparable frequency in the historical control data. A spontaneous origin is also assumed for the unclassified cartilage observations, which were recorded for fetuses of all dose-groups including the control. Character as well as distribution of all of these findings did not suggest a relation to treatment. In summary, there was no evidence of an adverse effect of Dimethylamine hydrochloride on fetal morphology at any dose level tested.

Thus, the oral administration of Dimethylamine hydrochloride to pregnant Wistar rats had no effect on morphology of offspring at any dose level tested (100; 300 and 1000 mg/kg bw/d). The recorded incidences did not suggest a treatment-relationship, but reflected the usual biological variation inherent in the strain of rats used for this experiment. In conclusion, the no observed adverse effect level (NOAEL) for maternal toxicity is 300 mg/kg bw/d based on decreased food consumption and salivation after treatment in the high dose dams (1000 mg/kg bw/d). The no observed adverse effect level (NOAEL) for prenatal developmental toxicity is 1000 mg/kg bw/d because there was no evidence of an adverse effect of the test compound on fetal morphology.

In a supporting study performed in mice (CD-1) by Guest and Varma (1991), maternal and fetal effects were investigated after administration of DMA as hydrochloric salt via intraperitoneal injection (11.3, 45.1, 112.7, 225.4 mg/kg bw, duration 17 days). The number of resorbed and dead fetuses were equally distributed across all doses of DMA, but the highest dose (225,4 mg/kg bw) caused a not significant increased number of dead fetuses and a significant higher number of resorptions. None of the amines (MMA, DMA, TMA) caused a significant increase in external, internal organ, or skeletal abnormalities, but all three possess a teratogenic potential in varying degrees. They reported a significant increase of mortality and number of dead fetuses at the highest dose of 225,4 mg/kg bw; a significant decrease of fetal body weight at 112,7 and 225,4 mg/kg bw. In vitro, all three methylamines produced concentration-dependent decreases in yolk-sac diameter, crown-rump length, head length, and fetal survival; developmental score and somite number also exhibited a similar concentration-dependent decrease. The effect of all the three methylamines was more marked on the head length than on crown-rump length and yolk-sac diameter. The development of yolk-sac circulation was severely affected at 112.7 mg/kg bw DMA. The colour of the yolk sac of DMA-treated embryos was paler than control and there appeared to be a decrease in flow rather than vascularization. The external appearance of the embryos was not affected by low concentrations of methylamines, but at higher concentrations (> 0,5 mM), there appeared a disproportionate retardation in the forelimb and branchial bar development relative to the development of other organs.All three methylamines produced concentration-dependent decreases in embryo RNA, DNA and proteins; the relative order of toxicity was the same as in vivo, namely TMA> DMA > MMA. DMA inhibits development of mouse embryos in culture and acts as endogenous teratogen under certain conditions. From the above, it was considered that reproductive/developmental toxicity NOAEL is 112.7 mg/kg/day for delivered pups.

Inhalation developmental toxicity study in second species (rabbits)

The objectives of the study were to determine the potential of the test substance, dimethylamine, to induce developmental toxicity after maternal exposure from implantation to 1 day priod to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect concentration (NOAEC) for maternal toxicity and developmental toxicity (WIL Reseach, 2016). Dimethylamine (DMA) was administered via whole-body inhalation exposure to 3 groups of 24 time-mated female New Zealand White [Hra:(NZW)] rabbits for 6 hours per day from gestation days 7 through 28. Target exposure concentrations were 50, 100, and 250 ppm for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) composed of 24 time-mated females was exposed to humidified, filtered air on a comparable regimen. Due to limitation of exposure chamber size, only 12 rabbits could be placed into each exposure chamber; therefore, this study was conducted in 2 phases, with 12 animals/group in each phase. The females were approximately 6 months of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. The eyes of all animals were examined for ocular irritation on gestation day 3or 4 (day of animal receipt) and gestation day 28. On gestation day 29, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

All females in the control and test substance-exposed groups survived to the scheduled necropsy on gestation day 29.

Rales were noted in 250 ppm group at the daily examinations and at 1‑2 hours following exposure throughout the exposure period. An increase in the incidence of elevated head was also noted in the 250 ppm group at the mid-point of exposure throughout the exposure period. Clear material around the eyes, mouth, and nose were noted at the daily examinations, approximate mid-point of each daily exposure, and/or 1‑2 hours following exposure for the 250 ppm group throughout the exposure period compared to the control group. The aforementioned clinical observations at 250 ppm were considered test substance-related and adverse. In addition, test substance-related clear material around the nose was noted for 50 and 100 ppm groups at 1‑2 hours following exposure throughout the exposure period. In the absence of other signs of toxicity at 50 and 100 ppm, and because the material findings did not persist to the daily examinations, they were not considered adverse at these exposure levels.

Test substance-related mean body weight losses were noted in the 250 ppm group during gestation days 7-10 compared to mean body weight gains in the control group. Corresponding lower mean food consumption was noted in this group generally throughout the exposure period. The initial mean body weight losses in the 250 ppm group were not of sufficient magnitude to result in lower mean body weight at the end of the exposure period. A mean net body weight loss was noted in the 250 ppm group. Mean maternal body weights, net body weights, and gravid uterine weights in all test substance-exposed groups and mean body weight gains and net body weight changes in the 50 and 100 ppm groups were unaffected by test substance exposure.

There were no remarkable ocular or macroscopic findings in any exposure group.

Intrauterine growth, survival, and fetal morphology (external, visceral, and skeletal) were unaffected by test substance exposure at all exposure levels.

Based on the higher incidence clinical observations (rales, elevated head, and clear material findings), mean body weight losses, and lower food consumption in the 250 ppm group, an exposure level of 100 ppm was considered to be the no‑observed‑adverse‑effect concentration (NOAEC) for maternal toxicity. There were no test substance-related effects on intrauterine growth, survival, and fetal morphology at any exposure concentration; therefore, the NOAEC for embryo/fetal development was 250 ppm when dimethylamine was administered via whole‑body inhalation exposure for 6 hours per day from gestation days 7 through 28 to time-mated New Zealand White rabbits.

The concentration in ppm was converted into mg/m³ according to the formula presented in the section 3.1.2.3.2. of ECHA guidance on the Application of Regulation (EC) No. 1272/2008 (2015):

mg/m³ = (MW x ppm)/ 24.45, where MW is molecular weight and 24.45 mg/m³ is the volume of ideal gas by 25°C. MW of Dimethylamine is 45.08 g/mol.

mg/m³ = (45.08 x 250 ppm) / 24.45 = 461 mg/m³.


Justification for selection of Effect on developmental toxicity: via oral route:
The only developmental toxicity study by oral route available.

Justification for selection of Effect on developmental toxicity: via inhalation route:
Recent developmental toxicity study in second species (rabbit).

Justification for selection of Effect on developmental toxicity: via dermal route:
No study is selected since a recent inhalation developmental toxicity study in second species (rabbit) is available. NOAEL for dermal route can be estimated using route-to-route extrapolation procedure.

Justification for classification or non-classification

Since no developmental toxicity to fetuses of rats have been shown up to a concentration of 1000 mg DMA hydrochloride/ kg bw/day, DMA is considered not to possess an embryo/fetotoxicity including teratogenic potential.

Also the results obtained from the dams, indicate that the maternal toxicity starts at doses above 300 mg/kg bw/day dose level below that of developmental toxicity. Therefore, the classification is not warranted according to the criteria of EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulations No 1272/2008.