Trimethylamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study; no data regarding GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Combined repeat dose and reproductive/developmental toxicity screening test of trimethylamine by oral administration in rats.

Author:

Takashima, H., Kuwagata, M., Miyahara, T., Katou, H., Seki, T., Shindou, T., Marumo, H., Horiuchi, S., Inada, H., Saegusa, K., Anjou, T., and Ichihara, S.

Year:

2003

Bibliographic source:

Hatano Research Institute, Food and Drug Safety Center, Japan.

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

No data on detailed clinical observation, sensory reactivity to stimuli, assessment of grip strength, and motor activity

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870-3650

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Trimethylamine

EC Number:

200-875-0

EC Name:

Trimethylamine

Cas Number:

75-50-3

Molecular formula:

C3H9N

IUPAC Name:

N,N-dimethylmethanamine

Details on test material:

CAS 75-50-3 (trimethylamine), 30.8% trimethylamine solution, SOURCE: Mitsubishi Gas Chemical Company, Inc. (Niigata; lot # M381012)
The solution contained < 10 ppm of dimethylamine as impurities. Stable for at least 8 days under refrigerated and shielded conditions with a concentration of between 0.08 and 10w/v%.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

-Age: 9 week old

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

42 days; Females: 2 weeks prior to breeding, continuing through breeding (2 weeks), gestation (3 weeks), lactation (4 days), and until the day of necropsy (test day 40 or 54).

Details on mating procedure:

Male/female per cage maximum for 2 weeks beginning evening of dosing day  15. Mating confirmed by existence of sperm in the vaginal plug  and vaginal smear every morning.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Once daily

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

(in water)

Doses / concentrations 2

Dose / conc.:

8 mg/kg bw/day

Remarks:

(in water)

Doses / concentrations 3

Dose / conc.:

40 mg/kg bw/day

Remarks:

(in water)

Doses / concentrations 4

Dose / conc.:

200 mg/kg bw/day

Remarks:

(in water)

No. of animals per sex per dose:

13/sex/dose group

Control animals:

yes, concurrent vehicle

Details on study design:

METHOD FOLLOWED: OPPTS and OECD Combined Repeated Dose Toxicity  Study with the Reproduction/Developmental Toxicity Screening Test DEVIATIONS FROM GUIDELINE: No data on detailed clinical observation, sensory reactivity to stimuli, assessment of grip strength, and motor activity.  STATISTICAL METHODS: Fisher's Exact Test- mating and conception rate,  Mann-Whitney U Test (2-tailed) and Fisher's Exact Test (1-tailed)-  histopathological examinations, Dunnett's Multiple Comparison Test  (significance level=5%)- body weight, food consumption, haematology, clinical chemistry and organ weights

Examinations

Parental animals: Observations and examinations:

- General condition was observed at least once a day during breeding and at least twice a day before and after dosing over the administration period. Body weights for males were determined on days 1, 7, 14, 21, 28, 35, 42 and on the day of necropsy. - Body weights were determined for all females on days 1, 7, 14. Females who took time before mating were weighed on days 35 and 42. Females who copulated were weighed on pregnancy days 0, 7, 14 and 20. Females who delivered were weighed on nursing days 0, 4, and on the day of necropsy. Females who copulated but did not deliver were weighed on the equivalent of pregnancy day 25 (day of necropsy).
- Food consumption was measured on days 1, 7, 13, 29, 35 and 41 for males, and on days 1, 7, and 13 for all females. Females with unconfirmed copulation were measured for food consumption on days 29, 35 and 41.
- Observation of delivery: With all cases where delivery was confirmed, the pregnancy period (number of days from pregnancy day 0 to delivery day) was calculated and the birth rate ((number of females who delivered live pups/number of animals conceived) x 100) for each group was found.
- Urinalysis was conducted on 5 rats/sex/dose level at week 6.
- Haematology and clinical chemistry: Males prior to necropsy and on the following day after day 42 administration; Females prior to necropsy: females who delivered-following nursing day 4, females who mated but did not deliver-equivalent of pregnancy day 25, and females who did not mate- following day 54 of administration

Oestrous cyclicity (parental animals):

-Oestrus Cycle: Oestrus cycle was observed from the vaginal smear and  the mean number of days of mating season was calculated..

Litter observations:

- Weights of Fetus: Pup weight was recorded on day 0 and 4 of lactation.
- Number of delivered pups: The number of delivered pups (live pups + stillborns) was counted on nursing day 0, and the delivery rate ((number of delivered pups/number of implantation traces) x 100) and the live birth rate ((number of delivered live pups/number of implantation traces) x 100) were calculated. With the delivered pups, external malformation and the sex were checked and the sex ratio ((number of male live pups/number of female live pups) x 100) was calculated.
- Number of dead pups: The number of dead pups was checked daily, and the birth rate ((number of delivered live pups/number of delivered pups) x 100) and the viable rate of new-born pups on day 4 ((number of live pups on nursing day 4/number of live pups on nursing day 0) x 100) were calculated. Dead pups were autopsied and abnormality and internal organs were observed.

Postmortem examinations (parental animals):

- Haematology and clinical chemistry: Males prior to necropsy and on the following day after day 42 administration; Females prior to necropsy: females who delivered - following nursing day 4, females who mated but did not deliver - equivalent of pregnancy day 25, and females who did not mate - following day 54 of administration

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):
- Macroscopic: organ weights: brain, heart, thymus, liver, kidneys, spleen, adrenals, testes and epididymides; pups were autopsied on day 4 and external and internal organs observed; with females, ovaries and uteri were extracted, the pregnancy corpora lutea number of the ovary was counted under the stereoscopic microscope, the implantation number of the uterus was counted, and the implantation rate ((implantation number/pregnancy corpora lutea number) x 100) was calculated.
- Microscopic: 5 animals/sex/control and high dose group- brain, pituitary gland, spinal cord, digestive tract, liver, kidneys, adrenal, spleen, heart, thymus, thyroid gland, trachea, lung, bladder, mesenteric lymph nodes, lower jaw lymph nodes, sciatic nerves, thigh bone marrow, sperm and prostrate ventral lobes of all males and vagina, ovaries and uteri of all females; also testes, epididymides, ovaries and stomachs found to be abnormal during pathologic examinations were all examined histopathologically

Postmortem examinations (offspring):

- Macroscopic: pups were autopsied on day 4 and external and internal organs observed.

Statistics:

Fisher's Exact Test- mating and conception rate,
Mann-Whitney U Test (2-tailed) and Fisher's Exact Test (1-tailed)- histopathological examinations,
Dunnett's Multiple Comparison Test (significance level = 5%)- body weight, food consumption, haematology, clinical chemistry and organ weights

Reproductive indices:

- Copulation: When mating was confirmed that day was reckoned as pregnancy day 0. From the results of copulation, mating rate ((number of animals mated/number of animals cohabited) x 100), conception rate ((number of animals conceived/number of animals mated) x 100), number of days needed for mating from the start of cohabitation and the number of times of oestrus which recurred during that time were calculated.
- Observation of delivery: With all cases where delivery was confirmed, the pregnancy period (number of days from pregnancy day 0 to delivery day) was calculated and the birth rate ((number of females who delivered live pups/number of animals conceived) x 100) for each group was found.

Offspring viability indices:

- Number of delivered pups: The number of delivered pups (live pups + stillborns) was counted on nursing day 0, and the delivery rate ((number of delivered pups/number of implantation traces) x 100) and the live birth rate ((number of delivered live pups/number of implantation traces) x 100) were calculated. With the delivered pups, external malformation and the sex were checked and the sex ratio ((number of male live pups/number of female live pups) x 100) was calculated.
- Number of dead pups: The number of dead pups was checked daily, and the birth rate ((number of delivered live pups/number of delivered pups) x 100) and the viable rate of new-born pups on day 4 ((number of live pups on nursing day 4/number of live pups on nursing day 0) x 100) were calculated. Dead pups were autopsied and abnormality and internal organs were observed.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Clinical signs prior to death (200 mg/kg/day):
Day 25 male, showed salivation, emaciation, abnormal breathing noise and dyspnoea from administration day 19, and vulval periphery fur soil and loose passage were observed from the day before the death.
Day 42 male, showed salivation, emaciation, abnormal breathing noise, dyspnoea, a drop in body temperature, faded auricle, tottering and brown soil around the nose were observed from administration day 10, although discontinuously.
The female was observed with salivation and abnormal breathing noise sporadically from administration day 11.
- Clinical signs in surviving animals: 200 mg/kg/day: Males- salivation 10/13, abnormal breathing noise 3/13, and decreased contact response 1/13; Females-salivation 10/13, abnormal breathing noise 3/13, and emaciation 1/13. 40 mg/kg/day: no abnormalities

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality and time to death: 1 male given 200 mg/kg/day died on day 25, 1 male given 200 mg/kg/day died on day 42 and 1 female died on pregnancy day 22 (administration day 38)

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males - No significant differences in body weights at 200 mg/kg/day; however, body weight gains were decreased when compared to controls. There were no significant differences in body weight or body weight gains in males administered 40 mg/kg/day.
Females - No significant differences in body weight or body weight gains. There was no effect of trimethylamine administration on body weights and food consumption of the females and on organ weights, urine examination and haematological examination results in the males and females

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

There was no effect of trimethylamine administration on haematological examination results in the males and females.

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Description (incidence and severity):

There was no effect of trimethylamine administration on haematological examination results in the males and females, besides a significant increase in urea nitrogen at 40 mg/kg bw, which was not considered to be adverse.

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

- Mortality and time to death: 1 male given 200 mg/kg/day died on day 25, 1 male given 200 mg/kg/day died on day 42 and 1 female died on pregnancy day 22 (administration day 38)
- Clinical signs prior to death (200 mg/kg/day): Day 25 male, showed salivation, emaciation, abnormal breathing noise and dyspnea from administration day 19, and vulval periphery fur soil and loose passage were observed from the day before the death. Day 42 male, showed salivation, emaciation, abnormal breathing noise, dyspnoea, a drop in body temperature, faded auricle, tottering and brown soil around the nose were observed from administration day 10, although discontinuously. The female was observed with salivation and abnormal breathing noise sporadically from administration day 11.
- Clinical signs in surviving animals: 200 mg/kg/day: Males- salivation 10/13, abnormal breathing noise 3/13, and decreased contact response 1/13; Females-salivation 10/13, abnormal breathing noise 3/13, and emaciation 1/13. 40 mg/kg/day: no abnormalities
- Body weights:
Males- No significant differences in body weights at 200 mg/kg/day; however, body weight gains were decreased when compared to controls. There were no significant differences in body weight or body weight gains in males administered 40 mg/kg/day.
Females - No significant differences in body weight or body weight gains.
There was no effect of trimethylamine administration on body weights and food consumption of the females and on organ weights, urine examination and haematological examination results in the males and females
- Clinical chemistry: Significant increase in urea nitrogen at 40 mg/kg/day

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: systemic toxicity

2

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Description (incidence and severity):

number of dead pubs was not influenced significantly by Trimethylamine administration

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio was not influenced

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

- Number of delivered pups (live pups + stillborns) was counted
- delivery rate ((number of delivered pups/number of implantation traces) x 100) was calculated
- the live birth rate ((number of delivered live pups/number of implantation traces) x 100) was calculated.
With the delivered pups, external malformation and the sex were checked and the sex ratio ((number of male live pups/number of female live pups) x 100) was calculated.
- Number of dead pups was checked daily
- the birth rate ((number of delivered live pups/number of delivered pups) x 100) was calculated
- the viable rate of new-born pups on day 4 ((number of live pups on nursing day 4/number of live pups on nursing day 0) x 100) was calculated.
- Dead pups were autopsied and abnormality and internal organs were observed.

from all of the above: no embryo-/fetotoxicity was observed (no effect was found on the viability of the delivered pups, sex ratio, body weight and form)

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

organ weights and organ / body weight ratios

gross pathology

Remarks on result:

other: no effect was found on the viability of the delivered pups, sex ratio, body weight and form

Remarks:

.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Daily oral administration of trimethylamine by gavage resulted in the death of two males and 1 female administered 200 mg/kg/day. Abnormal breathing noise, salivation immediately after the administration, ulcers and inflammatory changes in the stomach and intestinal tracts, squamous hyperplasia and oedema in submucosa were observed in both males and females in the 200 mg/kg/day group. An inhibition tendency in body weight increase, decrease in food consumption, total protein concentration and albumin concentration were also observed in the males in the same group.
There was no effect of trimethylamine administration on body weights and food consumption of the females and on organ weights, urine examination and haematological examination results in the males and females. The slight increase in urea nitrogen observed at 40 mg/kg bw is not considered to be adverse. Therefore it was inferred that the general toxicological NOAEL (No Observed Adverse Effect Level) is 40 mg/kg/day and the reproductive/developmental toxicity NOEL is 200 mg/kg/day for both males and females and also for delivered pups.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed by Takashima et.al. in 2003. It was performed in accordance to OECD guideline 422. Male and female rats (Sprague-Dawley, 13 animals/sex/dose group) were used for oral gavage of trimethylamine via water. The exposure time was 42 days, one dose daily. The dose groups were 0, 8, 40, and 200 mg/kg/day, respectively. Each one male given 200 mg/kg/day died on administration day 25, 38, and 42 respectively. Day 25 male, showed salivation, emaciation, abnormal breathing noise and dyspnoea from administration day 19, and vulval periphery fur soil and loose passage were observed from the day before the death. Day 42 male, showed salivation, emaciation, abnormal breathing noise, dyspnoea, a drop in body temperature, faded auricle, tottering and brown soil around the nose were observed from administration day 10, although discontinuously. The female was observed with salivation and abnormal breathing noise sporadically from administration day 11.

Clinical signs in surviving animals: 200 mg/kg/day: Males- salivation 10/13, abnormal breathing noise 3/13, and decreased contact response 1/13; Females-salivation 10/13, abnormal breathing noise 3/13, and emaciation 1/13. 40 mg/kg/day: no abnormalities - Body weights: Males- No significant differences in body weights at 200 mg/kg/day; however, body weight gains were decreased when compared to controls. There were no significant differences in body weight or body weight gains in males administered 40 mg/kg/day. Females - No significant differences in body weight or body weight gains. There was no effect of trimethylamine administration on body weights and food consumption of the females and on organ weights, urine examination and haematological examination results in the males and females. There was a significant increase in urea nitrogen at 40 mg/kg/day, which however is not considered to be adverse.

Maternal toxicity did not occur at 200 mg/kg, and no embryo-/fetotoxicity was observed. No abnormality which can be considered the effects of trimethylamine administration was observed in the reproductive organs, and no effect on the observation results of oestrous cycle was found. Also no effect was found on the mating rate, conception rate, the birth rate, pregnancy period, and nursing state, the number of corpora lutea, the number and rate of implantation, the viability of the delivered pups, sex ratio, body weight and form. From the above, it was considered that reproductive/developmental toxicity NOEL is 200 mg/kg/day for both males and females and also for delivered pups.