Dimethylamine

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Corrigendum to ECC Directive 2004/73/EC, Part B

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dimethylamine hydrochloride

IUPAC Name:

Dimethylamine hydrochloride

Constituent 2

Reference substance name:

Dimethylammonium chloride

EC Number:

208-046-5

EC Name:

Dimethylammonium chloride

Cas Number:

506-59-2

Molecular formula:

C2H7N.ClH

IUPAC Name:

N-methylmethanaminium chloride

Details on test material:

- Name of test material (as cited in study report): Dimethylamine hydrochloride
- CAS-No.: 506-59-2
- Test substance No.: 07/0090-1
- Analytical purity: 99.6%
- Homogeneity: Given
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.: JB132
- Expiration date of the lot/batch:
- Stability under test conditions: at least 10 days, (see below)
- Storage condition of test material: room emperature

ADDITIONAL TEST SUBSTANCE INFORMATION
Date of production: 15 Oct 2007
Expiry date: Unknown
Physical state/Appearance: Solid/white
Storage conditions: Room temperature, store under N2

For the preparation of the solutions, appropriate amounts of the test substance was weighed in graduated measuring flasks, topped up with drinking water and subsequently intensely shaken.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system 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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

nominal in water

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

nominal in water

Doses / concentrations 3

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

Examinations

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)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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).

Effect levels (maternal animals)

1

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

2

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

Results (fetuses)

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

Effect levels (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

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

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).

Applicant's summary and conclusion

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.