Trimethylammonium chloride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The route of exposure (i.p.) is not standard. Beside that an acceptable, well-documented publication which meets basic scientific principles.

Data source

Reference

Reference Type:

publication

Title:

Developmental toxicity of methylamines in mice.

Author:

Guest, I., and Varma, D.

Year:

1991

Bibliographic source:

Journal of Toxicology and Environmental Health 32: 319-330.

Materials and methods

Principles of method if other than guideline:

Exploration if chronic administration of methylamines can cause reproductive toxicity using pregnant CD-1mice and CD-1 mouse embryos in culture as experimental models.

GLP compliance:

not specified

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Trimethylammonium chloride

EC Number:

209-810-0

EC Name:

Trimethylammonium chloride

Cas Number:

593-81-7

Molecular formula:

C3H9N.ClH

IUPAC Name:

N,N-dimethylmethanaminium chloride

Details on test material:

CAS 593-81-7 (trimethylamine hydrochloride), purity not specified, obtained from Sigma Chemical Company (USA)

Specific details on test material used for the study:

no details given

Test animals

Species:

mouse

Strain:

CD-1

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, St. Constant, Quebec
- Weight at study initiation: 20-25 g
- Housing: Plexiglas cages with heat-treated wood chip bedding
- Diet (e.g. ad libitum): laboratory mouse chow pellets ad libitum
-Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-25 °C
- Humidity (%):50-70 %
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

other: 0.9 % saline

Details on exposure:

intraperitoneal injections, once a day between 8:00 and 9:00 a.m.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Details on mating procedure:

MATING PROCEDURES: 
not reported;
Day 0 of gestation was the day of mating.
mice bought already mated

Duration of treatment / exposure:

day 1-17 of gestation

Frequency of treatment:

daily

Duration of test:

until day 18 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

15 mg/kg bw/day (nominal)

Remarks:

= 0.25 mmol/kg bw

Doses / concentrations 2

Dose / conc.:

59 mg/kg bw/day (nominal)

Remarks:

= 1.0 mmol/kg bw/day

Doses / concentrations 3

Dose / conc.:

148 mg/kg bw/day

Remarks:

= 2.5 mmol/kg bw/day

Doses / concentrations 4

Dose / conc.:

295 mg/kg bw/day

Remarks:

= 5 mmol/kg bw/day

No. of animals per sex per dose:

4-11/treatment, 29 controls (used for 3 studies)

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female
Duration of test: day 0-18 of gestation

Examinations

Maternal examinations:

- Mortality/clinical observations/body weight (gain): yes, frequency not indicated
- Maternal and fetal body weight, mortality, resorptions, litter size, and placental weight were recorded in the in-vivo part of the study

Ovaries and uterine content:

- Examination of uterine content: placental weight, resorptions, litter size: yes
- Examination of the uterus for obvious sign of implantations (uteri were stained in 10 % ammonium sulfide solution to identify implantation site)

Fetal examinations:

- Examination of fetuses: viability, body weight, visceral and skeletal examination: yes
Pubs weight for each female were calculated by dividing the sum of body weights of all live pups in a litter by the number of live pubs in the litter
Mean pub weight for each treatment group was based on sum of mean pup weight for each female divided by the number of females in the group
Fetuses were randomly placed either in Bouin solution for visceral examination by the freehand razor sectioning technique or in 95 % ethanol for the skeletal examination by an alizarin red S staining technique.

Statistics:

Student's t-test, ANOVA followed by Bonferroni test

Indices:

no data

Historical control data:

no data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In treatment groups at 148 and 295 mg/kg bw contractions of the muscles at the injection site (~10 minutes after dosing). 
At 148 and 295 mg/kg bw ataxia, rapid shallow breathing, nasal discharge and tremors lasting from ~3 minutes after dosing until 10 minutes thereafter (recovery within 20 minutes).

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

5 of 11 pregnant females at 295 mg/kg bw died

Body weight and weight changes:

no effects observed

Description (incidence and severity):

All treated animals were within control ranges

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

- Number of resorbed and dead fetuses were equally distributed across all doses of TMA, but the highest dose (5 mmol/kg bw) caused a not significant increased number of dead fetuses and a significant higher number of resorptions.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Number of resorptions (mean)/female: at 0, 15, 59, 148 and 295 mg/kg bw: 0.66, 0.33, 0.25, 0.44 and 1.38
the highest dose (5 mmol/kg bw) caused a significant higher number of resorptions.

Dead fetuses:

no effects observed

Description (incidence and severity):

- Number of resorbed and dead fetuses were equally distributed across all doses of TMA, but the highest dose (5 mmol/kg bw) caused a not significant increased number of dead fetuses and a significant higher number of resorptions.

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

all females were pregnant

Other effects:

effects observed, treatment-related

Description (incidence and severity):

decreased placental weight (in the two high dose groups)
Placental weight (mean): at 0, 15, 59, 148 and 295 mg/kg bw: 109, 118, 116, 103 and 98 mg

Details on maternal toxic effects:

Maternal toxic effects at the highest dose.

Details on maternal toxic effects:
MATERNAL TOXIC EFFECTS BY DOSE LEVEL: 
- Mortality: 5 of 11 pregnant females at 295 mg/kg bw died
- Body weight: all treated animals were within control ranges
- Clinical signs: in treatment groups at 148 and 295 mg/kg bw contractions of the muscles at the injection site (~10 minutes after dosing). At 148 and 295 mg/kg bw  ataxia, rapid shallow breathing, nasal discharge and tremors lasting from ~3 minutes after dosing until 10 minutes thereafter (recovery within 20 minutes).

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

59 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

- Litter size (weights (mg)): at 0, 15, 59, 148 and 295 mg/kg bw: 10(1.4), 10(1.4), 8(1.4), 11(1.1) and 11(1.0)

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

- Number dead (mean)/litter: at 0, 15, 59, 148 and 295 mg/kg bw: 0.83, 1.11, 1.25, 0.56, 1.63
- Number of resorbed and dead fetuses were equally distributed across all doses of TMA, but the highest dose (5 mmol/kg bw) caused a not significant increased number of dead fetuses and a significant higher number of resorptions.

Changes in sex ratio:

not specified

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

- Litter size (weights (mg)): at 0, 15, 59, 148 and 295 mg/kg bw: 10(1.4), 10(1.4), 8(1.4), 11(1.1) and 11(1.0)

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Description (incidence and severity):

None of the amines (MMA, DMA, TMA) caused a significant increase in external, internal organ, or skeletal abnormalities.

Skeletal malformations:

no effects observed

Description (incidence and severity):

None of the amines (MMA, DMA, TMA) caused a significant increase in external, internal organ, or skeletal abnormalities.

Visceral malformations:

no effects observed

Description (incidence and severity):

None of the amines (MMA, DMA, TMA) caused a significant increase in external, internal organ, or skeletal abnormalities.

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
- Number pregnant per dose level: all females were pregnant
- Number of resorptions (mean)/female: at 0, 15, 59, 148 and 295 mg/kg bw: 0.66, 0.33, 0.25, 0.44 and 1.38
- Placental weight (mean): at 0, 15, 59, 148 and 295 mg/kg bw: 109, 118, 116, 103 and 98 mg

FETAL DATA: 
- Litter size (weights (mg)): at 0, 15, 59, 148 and 295 mg/kg bw: 10(1.4), 10(1.4), 8(1.4), 11(1.1) and 11(1.0)
- Number dead (mean)/litter: at 0, 15, 59, 148 and 295 mg/kg bw: 0.83, 1.11, 1.25, 0.56, 1.63
- Abnormalities: none observed

- Number of resorbed and dead fetuses were equally distributed across all doses of TMA, but the highest dose (5 mmol/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.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

295 mg/kg bw/day

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

NOAEL

Effect level:

59 mg/kg bw/day

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

STATISTICAL RESULTS: significant increase of mortality and number of dead fetuses at 295 mg/kg bw; significant decrease of fetal body weight at 148 and 295 mg/kg bw.

TABLE 3. Effects of Trimethylamine on Pregnancy Outcome in CD-1 Mice
Variables	Dose (mmol/kg/d, ip, d 1-17 of gestation)
	0	0.25	1.0	2.5	5.0
Animals (n)	29	9	4	11	11
Day 1 body weight (g)	24.1 ± 0.4	23.4 ± 0.5	23.1 ± 0.4	23.4 ± 0.5	28.2 ± 0.9
Day 18 body weight (g)	48.4 ± 1.1	50.0 ± 1.5	46.4 ± 3.0	45.7 ± 0.9	48.6 ± 2.8
Dams dead (n)	0	0	0	0	5^a
Dead fetuses/litter	0.83 ± 0.14	1.11 ± 0.35	1.25 ± 0.75	0.56 ± 0.24	1.63 ± 0.42^a
Resorptions/litter	0.66 ± 0.13	0.33 ± 0.17	0.25 ± 0.25	0.44 ± 0.18	1.38 ± 0.56
Litter size (n)	10 ± 1	10 ± 0	8 ± 2	11 ± 1	11 ± 1
Fetal body weight (g)	1.4 ± 0.03	1.4 ± 0.1	1.4 ± 0.04	1.1 ± 0.08^a	1.0 ± 0.06^a
Placental weight (mg)	109 ± 2	118 ± 5	116 ± 9	103 ± 4	98 ± 5
ARI	1.6 ± 0.4	1.0 ± 0.3	1.5 ± 0.4	4.3 ± 1.1^a	8.0 ± 0.8^a
Note.Mice were killed on d 18 of gestation; fetal and placental weights represent mean of weights per litter; ARI (adverse reproductive index) represents cumulative maternal and fetal toxicity in arbitrary units. Data are mean ± SE. ^aSignificantly (p < .05) greater than the corresponding control value.

 

In vitro studies: 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 severly affected at 0.25 mM DMA and TMA. The color of the yolk sac of DMA- and TMA-treated embryos was paler than control and there appeared to be a decrease in flow rather than vascularization.

The external appearance of embryos was not affected by low concentrations of methylamines. At higher concentrations (> 0.5 mM), there appeared to be a dispropotionate retardation in forelimb and branchial bar development relative to the development of other organs. All embryos were dorsally convex at 1 mM MMA or DMA, but only 33 % at a dose of 1 mM TMA.

The development of hearts was unaffected at concentrations up to 1 mM of all 3 methylamines and neuropores closed well up to concentrations of 0.75 mM.

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.

- all three methylamines possess teratogenic potential in varying degrees.

Applicant's summary and conclusion

Conclusions:

The NOAEL for maternal and teratogenicity was 59 or 295 mg/kg bw based on the absence of any adverse findings at this dose, and for fetotoxicity the NOAEL was 59 mg/kg bw based on the absence of any adverse findings at this dose.

Executive summary:

Reproductive toxicity

In a toxicity study in mice (Guest et al., 1991), the intraperitoneal trimethylamine hydrochloride administration TMA caused contractions of abdominal muscles in the proximity of injection sites at a dose level of 2.5 (148 mg/kg) and 5 mmol/kg (295 mg/kg). These contractions lasted for about 10 min after drug administration. Within 3 min after injection of 2.5 and 5 mmol/kg TMA, mice became ataxic, the breathing became shallow and rapid (not quantified), there was nasal discharge, and tremores developed. Animals responded to touch and the righting reflex was not lost. These effects lasted for approximately 10 min and if the animals did not die during this period complete recovery occurred within 20 min. Because trimethylamine exerts a gross toxicity in pregnant mice, a number of virgin mice of similar starting age were also used for comparison. A dose of 5 mmol/kg TMA caused death of 5 of 11 pregnant and 6 out of 6 virgin mice. The death of pregnant mice (pregnancy ascertained by necropsy) was not preceded by an effect on body weight. However, virgin females steadily lost weight (approximately 0.5 g/d); 4 animals died on day 6 of treatment and all had died by day 9. From the above, it was considered that for maternal toxicity NOAEL is 59 mg/kg bw/day for female mice. 

Developmental toxicity

A study performed by Guest et al. in 1991, dealt with the investigation of maternal or fetal effects after administration of TMA via intraperitoneal injection. Trimethylamine adversely affected fetal development in vivo. It caused a decrease in fetal weight without affecting maternal body weight gain. Because of a relatively greater decrease in fetal than placental weights, ratios of placental to fetal weights increased in mice treated with 148 mg/kg and 295 mg/kg TMA. The number of resorbed and dead fetuses were equally distributed across all doses of TMA, but the highest dose (295 mg/kg bw). 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 295 mg/kg bw (45 % of mice died); a significant decrease of fetal body weight at 148 and 295 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. TMA (applied as TMA-HCl) was the methylamine with the most distinct toxic effects (inhibition of embryo development in culture). The development of yolk-sac circulation was severly affected at 0.25 mM DMA nad TMA. The color of the yolk sac was paler than control and there appeared to be a decrease in flow rather than vascularization. Only 33 % of the embryos were dorsally convex at 1 mM TMA. The development of hearts was unaffected at concentration up to 1 mM of all 3 methylamines and neuropores closed well up to concentrations of 0.75 mM. 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. At the highest concentration used (1 mM), TMA caused a significant increase in the ratio of RNA to DNA and protein to DNA; these ratios were not altered by MMA or DMA.

In conclusion: TMA was toxic to mouse fetuses in utero, and all the three amines tested inhibited development of mouse embryos in culture. So, TMA acts as endogenous teratogen under certain conditions. The NOAEL for fetotoxicity is determined as 59 mg/kg bw, based on the number of fetal death and the reduced body weight gain.The NOAEL for teratogenicity is 295 mg/kg bw/day.