Methanaminium, N,N,N-trimethyl-, 2,2-dimethylpropanoate (1:1)

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2017 - June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Test guideline: GB/T 21766-2008

Deviations:

not specified

GLP compliance:

not specified

Remarks:

Chinese study. No GLP declaration. "The test was strictly conducted in accordance with the national standards and relevant SOP."

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

478-310-4

EC Name:

-

Cas Number:

53803-13-7

Molecular formula:

C9H21NO2

IUPAC Name:

Methanaminium N,N,N-trimethyl-, salt with 2,2-dimethylpropanoic acid

Test material form:

other: Liquid

Details on test material:

Test was performed using commercial product DABCO TMR-7 which is 50% solution of Methanaminium N,N,N-trimethyl-, salt with 2,2-dimethylpropanoic acid in 1,2-ethandiol, Batch #19847-85

Specific details on test material used for the study:

Physical description: White solid
Purity: >99.5%
Lot Number: Ten.17.812
Storage: Keep away from light at room temperature
Date received: 2017.6.12

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

The animals were housed in SPF barrier environment. The temperature of the experimental animal room was 22 -25°C and the relative humidity was 44 65 percent, the light/dark cycle was 12 hours. The used license number of the experimental animal room: SYXK(xiang)2017-0001. The rats were raised in the special cage or box. During the test, the animal bedding were replaced once a week and the cages replaced fortnightly, the drinking bottles were cleaned and sterilized once a week. Animals were raised individually in the cage during the pre-mating, post-mating, and gestation period. On day 14 of
pregnancy, the maternal rats were fed in the rearing tanks with the nesting bedding.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Sprague-Dawley rats were randomly assigned to four groups with 12 rats per sex in each group. Methanaminium, N,N,N-trimethyl-, salt with 2,2-dimethylpropanoic acid(1:1) was administered to the rats by gavage in dose of 4.9 mg, 9.8 mg and 19.6 mg/(kg bw.d). Male rats were exposed to test substances for 4 weeks, including the pre-mating period (2 weeks), mating period and after mating period; female rats including the pre-mating period(2 weeks), mating period, gestation and lactation period (4 days).

Details on mating procedure:

Mating male and female rats in the same cage with proportion of 1:1 after two weeks (14 days) of exposure, the vaginal plug was checked in the morning daily to determine the pregnancy.
The day when vaginal plug appeared was set as 0 day of pregnancy. Mating period is up to two weeks.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

The test substance for the three dose groups were administered daily by gavage. The volume was 10 mL/kg.bw. Male exposure duration was 4 weeks, 2 weeks in pre-mating
period and at least 2 weeks in post-mating period. The exposure of female rats lasted for the entire test cycle, including 2 weeks in pre-mating period, mating period, gestation period and 4 days after delivery (the day of execution).

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle Control Group

Doses / concentrations 2

Dose / conc.:

4.9 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

9.8 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

19.6 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 males and 12 females per dose group

Control animals:

yes, concurrent vehicle

Positive control:

No

Examinations

Parental animals: Observations and examinations:

Maternal rats were observed for clinical signs at least once a day. Recorded paramters behavioral changes, mogitocia, bradytocia, the time toxicity occurred, degree, duration, and the mortality rate.

Male and female rats were weighed before exposure, and then weighed once a week and at the end of the test. Maternal rats were weighed at the 0, 7th, 14th 20th days during gestation, the day of delivery and the 4th day of postpartum.
Litter weight and body weight of pups were weighed at the day of delivery and the 4th day of postpartum period.
Food consumption was measured once a week during the pre-mating period, gestation, and lactation.

Oestrous cyclicity (parental animals):

The gestation cycle was recorded from day 0 of conception, it was also necessary to observe whether mogitocia or bradytocia occurred during the period of partum.

Litter observations:

Each litter was examined after delivery to record the sex of pups, the number of stillbirths, live births, low weight pups and the presence of abnormalities. Any abnormal findings of offspring on physiology and behavior was recorded. The mortality of pups was recorded from day 0 to day 4 after delivery. The pups of death and pups sacrifice at the day 4 after delivery were examined for gross abnormalities.

Postmortem examinations (parental animals):

Male rats were sacrificed on day 28 after exposure, the maternal rats with offspring were sacrifieced on day 4 after delivery, and female rats without mating were sacrificed on day 24 after mating period. All of the parental animals were examined macroscopically for any abnormalities or pathological changes. In particular, the organs of the reproductive system were examoned: The testes and epididymides of all male adult animals were weighed. The number of implantation sites and corpora lutea were recorded.

Postmortem examinations (offspring):

Pups that died within 4 days after delivery or were sacrificedon day 4 after delivery were examined carefully for appearance of abnormalities.

Statistics:

Software package SPSS 13.0 was used to perform statistics. Chi-square test, one-way ANOVA or non-parametric statistics and Wilcoxon-Wilcox rank sum test are used for statistical analysis where appropriate.

Reproductive indices:

Histopathological examination was performed on the ovaries, testis and epididymis of the animals in the high dose group and the solvent control group, especially the stage of spermatogenesis and the cell structure of the testicular interstitium. The other preserved organs may be examined when necessary. Examination was to be extended to the animals of other dosage groups when changes are seen in the highest dose group.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

No obvious clinical signs were observed in the three dose groups after exposure.
Maternal rats in the high dose group and all of the maternal rats in other dose groups showed no obvious signs of toxicity during each period such as premating, mating, pregnancy and lact

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no obvious histopathological changes in the testicles, epididymis, ovaries and uterus of the animals in the three dose groups.

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no significant differences in offspring loss before implantation, after implantation or after the birth compared with the solvent control group (p>0.05).

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

19.6 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

1

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

19.6 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

testes

2

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

19.6 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

ovary

uterus

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

19.6 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

mortality

organ weights and organ / body weight ratios

gross pathology

Target system / organ toxicity (F1)

1

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

19.6 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

testes

2

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

19.6 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

ovary

uterus

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

19.6 mg/kg bw/day (nominal)

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

The average litter weight and body weight, the sex ratio (m/f) of pups at birth, the average litter weight and body weight, the average litter weight gain and body weight gain, the sex ratio (m/f), the survival rate, the incidence of appearance deformity, small stature and subcutaneous ecchymosis of pups on the 4th day after delivery showed no significant differences compared with the solvent control group (p>0.05).
There were no significant differences in offspring loss before implantation, after implantation or after the birth compared with the solvent control group (p>0.05).

No histological changes occurred on testis and epididymis of male rats and ovary and uterus of female rats in the high dose groups.

No reproductive and developmental toxicity was observed under the experimental conditions.

Executive summary:

In an OECD 421 guideline study, 96 Sprague-Dawley (SD) rats of 61-65 days old were randomly assigned to four groups with 12 rats per sex in each group. The test item was administered daily by gavage in doses of 0, 4.9 mg, 9.8 mg and 19.6 mg/(kg bw.d).

Male rats were exposed to test substances for 4 weeks, including the pre-mating period (2 weeks), mating period and after mating period; female rats including the pre-mating period(2 weeks), mating period, gestation and lactation period (4 days).

Male and female rats were mated after two weeks of exposure, the vaginal plug was checked in the morning daily to determine pregnancy. The day when vaginal plug appeared was set as 0 day of pregnancy. Mating period is up to two weeks. Behaviors such as mating, gestation,

parturition and lactation were observed and the toxic signs, body weight and food consumption were recorded. On day of parturition and day 4 after delivery, litter weight and body weight of pups were weighed. The number of live pups, dead pups, low weight pups, sex of pups, and any variations or malformations were recorded.

Rats were sacrificed after exposure and gross necropsy and histopathology were examined. Any changes of the reproductive organs were recorded, as were the number of implantations and corpus lutea.

No signs of clinical toxicity were observed in the parental male rats in the three dose groups and female rats in the low and medium dose group. No obvious toxic signs were observed in the female rats in the high dose group during the pre-mating, mating and gestation period, only one female gave birth to one malformed offspring with left forelimb defect, showed biting off pups and refused to breastfeeding. Otherwise, there were no obvious deleterious effect on the offspring and no damage to the physiological or reproductive function of the parental rats.

There were no treatment-related parental mortalities during the experiment. The weekly body weight and the total body weight gain, the weekly food consumption and the total food consumption, absolute and relative weights of testicle and epididymis of the parental male rats in the three dose groups were no significant differences compared with the solvent control group (p>0.05).

The weekly body weight and the total body weight gain, the weekly food consumption and the total food consumption of the female rats during the pre-mating period and gestation, the body weight, body weight gain and food consumption during the lactation period, in the dose groups during the pre-mating, gestation of the female rats in each dose group showed no significant differences compared with the solvent control group (p>0.05).

The average mating time and the mating rate of the rats in each dose group, the conception rate, the implantation rate, and the average duration of gestation period and the pregnant rate of the female rats in each dose group showed no significant differences compared with the solvent control group (p>0.05).

The average litter weight and body weight, the sex ratio (m/f) of pups at birth, the average litter weight and body weight, the average litter weight gain and body weight gain, the sex ratio (m/f), the survival rate, the incidence of appearance deformity, small stature and subcutaneous ecchymosis of pups on the 4th day after delivery showed no significant differences compared with the solvent control group (p>0.05).

There were no significant differences in offspring loss before implantation, after implantation or after the birth compared with the solvent control group (p>0.05). No histological changes occurred on testis and epididymis of male rats and ovary and uterus of female rats in the high dose groups. In conclusion, administration of the test materials elicited no reproductive and developmental toxicity under the experimental conditions.