Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study; no data regarding GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
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
Qualifier:
according to
Guideline:
other: OPPTS 870-3650
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 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
Remarks:
Doses / Concentrations:
0, 8, 40, 200 mg/kg/day (in water)
Basis:

No. of animals per sex per dose:
13/sex/dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Method: 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, hematology,  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.

Oestrous cyclicity (parental animals):
-Estrus Cycle: Estrus cycle was observed from the vaginal smear and  the mean number of days of mating season was calculated..
Litter observations:
-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):
-Urinalysis was conducted on 5 rats/sex/dose level at week 6.
-Hematology 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: testes and epididymides; 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- sperm and prostrate ventral lobes of all males and vagina, ovaries and uteri of all females; also testes, epididymides, ovaries found to be abnormal during pathologic examinations were all examined histopathologically.


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, hematology, 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  estrus 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
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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, dyspnea, 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 hematological examination results in the males and females
-Clinical chemistry: Significant increase in urea nitrogen at  40  mg/kg/day

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: systemic toxicity

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Mortality / viability:
not specified
Body weight and weight changes:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Histopathological findings:
not specified

Details on results (F1)

no details given

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 edema 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 hematological examination results in the males and females. Therefore it was inferred that the general toxicological NOAEL (No Observed Adverse Effect Level) is 40 mg/kg/day.
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 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, dyspnea, 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 hematological examination results in the males and females.There was a significant increase in urea nitrogen at 40 mg/kg/day.

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 estrous 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.