1,3-di-o-tolylguanidine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Prenatal developmental toxicity study of the basic rubber accelerator, 1,3-di-o-tolylguanidine, in rats.

Author:

Makoto Ema, Sakiko Fujii, Mariko Matsumoto, Akihiko Hirose and Eiichi Kamata

Year:

2006

Bibliographic source:

Reproductive Toxicology, 22(4), 672-6780.

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1,3-di-o-tolylguanidine

EC Number:

202-577-6

EC Name:

1,3-di-o-tolylguanidine

Cas Number:

97-39-2

Molecular formula:

C15H17N3

IUPAC Name:

1,3-di-o-tolylguanidine

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Co.
- Age at study initiation: males = 11 weeks, females = 10 weeks
- Weight at study initiation:males = 354-401 g, females = 202-246 g
- Fasting period before study: no data
- Housing: 4 during the acclimatation period, 2 during the mating, then individually
- Diet (e.g. ad libitum): free access to sterilized solid food CRF-1 (oriental Yeast Co. Ltd) from metal food dishes
- Water (e.g. ad libitum): sapporo municipal water, ad libitum
- Acclimation period: 5 days prior the mating

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-3°C
- Humidity (%):50+/-20%
- Air changes (per hr):10-15/h
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5% CMC-Na solution with 0.1% Tween 80 added

Details on exposure:

Administration volume = 5 ml/kg
The administration amount was set on the results of the 28 day repeat oral toxicity test and the simple reproductive toxicity study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the test substance in the administration solutions for all of the concentrations during the first and last preparations were analyzed with HPLC, Millenium 32 chromatography manager, digital balance, compact refrigerated centrifuge, desktop ultrasonic cleaner, pH meter.

Details on mating procedure:

- Impregnation procedure: cohoused
The vagical mucous was sampled and observations were made on the estrous cycle under a microscope, and then one males and one female were placed together fot one night just before estrous.
- M/F ratio per cage: 1/1
- Length of cohabitation: one night. Mating occurred over a span of 6 days.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

GD6-GD19

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24 females/dose

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS + DETAILS CLINICAL OBSERVATIONS: Yes. The presence of dead animals and general observations on all animals were conducted twice daily (before and after administration) during the administration period, and at a frequency of once a day during the period before administration was started, and on the day of the necropsy.
BODY WEIGHT: Yes, on day 1, 3 and 6-20 of gestation
FOOD CONSUMPTION : Yes, on day 1, 3, 6, 9 12, 15, 18 and 20 of gestation
WATER CONSUMPTION : no
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: Brain (cerebrum and cerebellum), pituitary gland, thymus, thyroid gland (left/right), epididymis (left/right), adrenals (left/right), spleen, heart, liver, lungs (including the trachea), kidneys (left/right), ovaries (left/right), uterus (fundus and cervix), vagina, mammary gland (right side) and places where visual abnormalities were noted (including the boundary areas with normal tissues).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of dead foetuses : Yes
- Number of live foetuses: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No data

Statistics:

See below

Indices:

Implantation rate, fetal survival rate, embryo/fetus death rate, sex ratio.
Rate of foetuses with external abnormalities, frequency of litters with foetuses with external abnormalities.
Rate of foetuses with internal abnormalities, frequency of litters with foetuses with internal abnormalities.
Progression of ossification, rate of skeletal variation in fetuses, rate of skeletal abnormalities in fetuses, frequency of litters with foetuses with skeletal variation, frequency of litters with foetuses with skeletal abnormalities

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):

During observation on the general conditions of maternal animals during the administration period, alopecia was noted on two animals each in the control group and the 10 mg/kg group. In the 20 and 40 mg/kg groups, 12 and 24 animals respectively experienced mydriasis between 10 and 50 minutes after administration, and a reduction in spontaneous locomotor activity was noted in 1 and 11 animals respectively. Also, the symptoms of lying prone (3 animals), bradypnea (2 animals) and tremors (2 animals) were noted in the 40 mg/kg group, and 4 animals (animal numbers 451, 463, 468 and 471) perished within 30 minutes after administration (days 8, 8, 19 and 7 of gestation). Among the surviving animals, these symptoms disappeared between 10 minutes and 2 hours after administration. As for other symptoms, there were 2 cases of salivation, 4 cases of soil in the perigenital fur and 2 cases of alopecia in the 40 mg/kg group. In the 20 mg/kg group, there were 2 cases of salivation, one case of soil in the perigenital fur and 3 cases of alopecia but there were no mortalities in the maternal animals.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No significant differences were noted in the mean body weights and revised body weights of maternal animals in the 10 mg/kg group when compared to the control group. In the 20 mg/kg group, significantly low values were noted in the body weights on day 20 of gestation (necropsy day). In the 40 mg/kg group, significantly low values were noted in the body weights and the revised body weights from day 8 of gestation (day 3 of administration) until day 20 of gestation (necropsy day).
No significant differences were noted in the mean weight gain of maternal animals in the 10 mg/kg group when compared to the control group. In the 20 mg/kg group, all of the values were significantly low values on days 0-10 of gestation as well as from day 0-12 to day 0-20. In the 40 mg/kg group, significantly low values were noted throughout the administration period from day 0-7 of gestation to 0-20 of gestation. Significantly low values were noted in the 20 and 40 mg/kg groups for weight gain during the administration period (days 6-20 of gestation).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No significant differences were noted in the mean food consumption amounts of maternal animals in the 10 and 20 mg/kg groups when compared to the control group. In the 40 mg/kg group, significantly low values were noted in the amount of food consumed during the administration period, on days 6-9, 9-12, 12-15, 15-18 and 18-20 of gestation.

Food efficiency:

not specified

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

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

see section "clinical signs"

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

During the necropsy performed on the maternal animals in the control group on day 20 of gestation, there was one case of situs inversus viscerum and two animals with alopecia, while there was one case of alopecia in each of the 10 and 20 mg/kg groups, and two cases of alopecia, two cases of thymus atrophy and one case of cystic kidney in the 40 mg/kg group.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Details on maternal toxic effects:

Pregnancy was confirmed in all of the females in each group of 24 that successfully copulated, and with the exception of the females in the 40 mg/kg group that died, surviving fetuses were confirmed in all of these pregnant females. Significantly low values were noted for the gravid uterus weights in the 40 mg/kg group. Significantly high values were noted in the implantation rate in the 10 mg/kg group. Significantly different values were not noted between the control group and any of the test substance administration groups for the number of corpus luteum or number of implantations.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Maternal abnormalities

Abnormalities:

not specified

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

No significant differences were noted for the number of dead fetuses, fetal death rate, number of surviving fetuses and fetal survival rate in the 10 and 20 mg/kg groups when compared to the control group. In the 40 mg/kg group, significantly high values were noted for the number of dead fetuses (both early stage and overall) and the fetal death rate while significantly low values were noted for the number of surviving fetuses and fetal survival rate.

Changes in sex ratio:

effects observed, treatment-related

Description (incidence and severity):

Significantly low values were noted in all of the test substance administration groups for the fetal sex ratio.

Changes in litter size and weights:

effects observed, treatment-related

Changes in postnatal survival:

effects observed, treatment-related

External malformations:

effects observed, treatment-related

Description (incidence and severity):

During the external examination conducted on the surviving fetuses, there were 8 and 31 animals with short phalanges in the 20 and 40 mg/kg groups, respectively (the group means calculated from the rate of fetal external abnormalities for each litter were 2.17% and 14.38%), 7 and 10 animals with short tails (1.84% and 4.37%). In the 40 mg/kg group, the rate of incidence of short phalanges and short tails and the frequency in litters had significantly high values when compared to the values in the control group. Statistically significant differences were not noted in the 20 mg/kg group but a trend of high values was evident. Significantly high values were seen for the overall rate of fetuses with external abnormalities and the overall frequency in litters in the 40 mg/kg group. Additionally, one case of cleft palate was noted in the 10 mg/kg group (0.28%) but there were no significant differences in the rate of fetuses with abnormalities and the overall frequency in litters when compared to the values in the control group.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Skeletal abnormalities in the 20 and 40 mg/kg groups manifested as missing tails, fusion or positional abnormalities were noted in 8 and 10 animals respectively (the group means calculated from the rate of fetal skeletal abnormalities for each litter were 4.17% and 8.56%). Missing or fused phalanges were noted in 5 and 18 animals respectively (2.60% and 16.02%). The rate of skeletal abnormalities in fetuses and the frequency in the litter exhibited significantly high values in the 40 mg/kg group when compared to those of the control group. Statistically significant differences were not noted in the 20 mg/kg group but a trend of high values was evident. In the 40 mg/kg group, fusion of the metacarpals/metatarsals and phalanges was noted in 2 animals (1.55%), missing or fused metacarpals was noted in 4 animals (3.38%), shortening of the tibia and fibula was noted in one animal (1.25%), and the rate of missing or fused metacarpals in fetuses and the frequency in the litter was significantly high. Thus, in the 20 and 40 mg/kg groups, significantly high values were noted for the overall rate of fetal skeletal abnormalities, and the 40 mg/kg group also had significantly high values for the overall frequency in the litter. Additionally, there was one animal (0.60% and 0.83%) in each of the 20 and 40 mg/kg groups with split cartilage of thoracic centrum, one animal (0.46%, 0.69% and 1.25%) in each of the 10, 20 and 40 mg/kg groups with fused cartilage of the cervical vertebral arches, and one animal (0.60%) in the control group with fused cartilage of the ribs. However, the rate of fetal skeletal abnormalities and the frequency in the litter for these did not exhibit significant differences between the test substance groups and the control group.
Skeletal variation was observed in all of the test groups as well as the control group. The type and the number of fetuses (the group means were calculated from the rate of skeletal variation for each litter) included 9 (4.94%), 12 (6.73%), 14 (8.06%) and 4 (2.89%) animals each in the control, 10, 20 and 40 mg/kg groups with short supernumerary ribs, 1 (0.60%) and 2 (1.43%) each in the control and 40 mg/kg group with unossified thoracic centrum, 2 (0.93%) and 1 (0.60%) each in the 10 and 20 mg/kg groups with bipartite ossification center of thoracic centrum, 1 (0.60% and 0.52%) each in the 10 and 20 mg/kg groups with wavy ribs, and 1 (0.60% and 1.25%) each in the 20 and 40 mg/kg groups with bipartitie ossification of sternebra. Additionally, there was one case (0.52%) of dumbbell ossification of the thoracic centrum in the 10 mg/kg group, 2 (1.43%) animals in the 40 mg/kg group with a short 13th rib, two (1.43%) animals in the 40 mg/kg group with sacralization of lumbar vertebra and one (0.83%) with asymmetry of sternebra. However, the rate of fetal skeletal variation and the frequency in the litter for these did not exhibit significant differences between the test substance groups and the control group.
Significant differences in the mean number for fetal vertebral ossification, thoracic centrum, and ossification of the metacarpals/metatarsals were not noted when the 10 and 20 mg/kg groups were compared with the control group. In the 40 mg/kg group, significantly low values were noted for ossification of the sacral and coccygeal vertebrae, thoracic centrum, and the number of ossified metacarpals and metatarsals.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

For abnormalities with internal organs and tissues, there was one case in both the control group and the 20 mg/kg group with microphthalmia (0.60% and 0.52%), one case in the control group with dilatation of the lateral ventricle (0.60%), and one animal in the 40 mg/kg group with undescended testis (0.83%). However, no significant differences were noted in either the rate of abnormalities in fetal internal organs and tissues or the frequency in the litter when compared to the values of the control group.
Variation in the internal organs and tissues were noted in all of the test groups, including the control group. The number of fetuses (group mean calculated from the rate of variation in fetal internal organs and tissues for each litter) and their classifications included thymic remnant in the neck in 13 (7.52%), 8 (4.63%), 12 (6.87%) and 17 (14.80%) animals in the 10, 20 and 40 mg/kg groups, respectively, as well as left umbilical artery in 1 (0.69%), 1 (0.60%) and 2 (1.63%) animals, respectively, and dilatation of renal pelvis in 2 (1.22%) and 2 (1.35%) animals in the control group and the 10 mg/kg group, respectively. However, no significant differences were noted in either the rate of variation in fetal internal organs and tissues or the frequency in the litter when compared to the values of the control group.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

No significant differences were noted in the fetal weights and placenta weights for both sexes in the 10 and 20 mg/kg groups when compared to the control group, but significantly low values were noted for both in the 40 mg/kg group.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

Remarks on result:

other: At 20 mg/kg bw/d: external malformations (short phalanges, short tails), skeletal malformations (missing tails, fusion or positional abnormalities).

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Table 1: Maternal findings in rats given DOTG on days 6-19 of pregnancy

 

Dose (mg/kg)	0 (control)	10	20	40
No. of rats	24	24	24	24
No. of pregnant rats	24	24	24	24
Initial body weight	256+/-13	256+/-13	256+/-13	256+/-13
No. of females showing clinical signs of toxicity
Death	0	0	0	4
Alopecia	2	2	3	2
Bradypnea	0	0	0	2
Decreased locomotor activity	0	0	1	11**
Mydriasis	0	0	12	24**
Prone position	0	0	0	3
Salivation	0	0	2	2
Soil of perigenital	0	0	1	4
Tremor	0	0	0	2
Bodyweight gain during pregnancy (g) a)
Days 0-6	40+/-8	39+/-8	40+/-8	39+/-8
Days 6-15	50+/-7	49+/-9	37+/-11**	23+/-10**
Days 15-20	77+/-9	77+/-9	71+/-10	47+/-16**
Days 0-20	167+/-17	165+/-21	148+/-24**	109+/-21**
Adjusted weight gain b)	88+/-15	87+/-19	77+/-15	49+/-17**
Food consumption during pregnancy (g/day) a)
Days 0-6	23+/-2	23+/-2	23+/-2	23+/-2
Days 6-15	26+/-2	26+/-2	24+/-3	20+/-3**
Days 15-20	28+/-2	28+/-3	26+/-2	22+/-3**
Days 0-20	25+/-2	26+/-2	24/-2	21+/-2**
Weight of gravid uterus (g) a)	79+/-10	78+/-11	72+/-15	59+/-10**

a) values are given as the mean +/-SD.

b) adjusted weight gain refers to maternal weight gain excluding the gravid uterus.

** significantly different from the control (p<0.01).

 

 

Table 2: Reproductive findings in rats given DOTG on days 6-19 of pregnancy

 

Dose (mg/kg)	0 (control)	10	20	40
No. of litters	24	24	24	20
No. of litter totally resorbed	0	0	0	0
No. of corpora lutea per litter a)	15.7+/-2.1	14.8+/-1.6	14.9+/-1.9	15.3+/-1.5
No. of implantations per litter a)	15.3+/-1.9	14.7+/-1.8	14.2+/-2.7	15.2+/-1.4
% preimplantation loss per litter b)	2.4	0.9	5.6	0.9
% postimplantation loss per litter b)	3.5	3.4	4.8	16.4**
No. of live fetuses per litter a)	14.8+/-1.9	14.2+/-2.1	13.7+/-2.9	12.6+/-1.9**
Sex ratio of live fetuses (male/female)	0.56	0.49*	0.46*	0.46*
Bodyweight of live fetuses (g) a) :
-male	3.64+/-0.17	3.72+/-0.18	3.59+/-0.24	3.19+/-0.31**
-female	3.42+/-0.16	3.53+/-0.25	3.41+/-0.18	3.03+/-0.26**
Placental weight (g) a)	0.47+/-0.04	0.47+/-0.03	0.50+/-0.16	0.40+/-0.04**

Significantly different from the control (*: p< 0.05 and ** : p<0.01).

           

a)     values are given as the mean +/-SD.

b)      (No. of preimplantation embryonic loss/ no. of corpora lutea) x 100

c)     (No of resorptions and dead fetuses/ no. implantations) x 100

Applicant's summary and conclusion

Conclusions:

Pregnant rats were given 1,3-di-o-tolylguanidine (DTG) by gavage at 0, 10, 20 or 40 mg/kg bw/day on days 6-19 of pregnancy and the pregnancy outcome was determined on day 20 of pregnancy. At 40 mg/kg bw/day, deaths were observed in four out of 24 females. The incidences of females showing mydriasis at 20 and 40 mg/kg bw/day and showing decreased locomotor activity at 40 mg/kg bw/day were significantly increased. Alopecia, bradypnea, prone position and tremor were also observed at 40 mg/kg bw/day. The maternal body weight gain at 20 and 40 mg/kg bw/day and food consumption at 40 mg/kg bw/day were significantly reduced. A significantly decreased weight of the gravid uterus, increased incidence of postimplantation loss, decreased number of live fetuses, and lowered weights of fetuses and placentae were found at 40 mg/kg bw/day. The incidences of the total number of fetuses with external malformations at 40 mg/kg bw/day and with skeletal malformations at 20 and 40 mg/kg bw/day were significantly increased. Significantly higher incidences of fetuses with brachydactyly and short tail and defects of caudal vertebrae, phalanges and metacarpals were observed at 40 mg/kg bw/day. Delayed ossification was also noted at 40 mg/kg bw/day. The data indicate that DTG is teratogenic at maternal toxic doses and the NOAELs of DOTG for maternal and developmental toxicity are 10 mg/kg bw/day in rats.

Executive summary:

Using doses of 0 (control group), 10, 20 and 40 mg/kg/day of N, N’-bis (2-methylphenyl) guanidine administered orally using a gastric probe, the presence of toxicity on fetal rats was studied in 24 Crj (SD) IGS female rats per group with confirmed copulation from day 6 to day 19 of gestation.

During observations on the general conditions of the maternal animals, changes relating to administration of the test substance included mydriasis occurring 10 minutes to 50 minutes after administration in half of the animals in the 20 mg/kg group and all of the animals in the 40 mg/kg group. In the 40 mg/kg group, a reduction in spontaneous locomotor activity was also noted in approximately half of the animals. Additionally, four animals observed with the symptoms of lying prone, bradypnea and tremors died within 30 minutes after administration. The surviving animals recovered from these symptoms from about 10 minutes to two hours after administration. During the necropsy, changes believed to be related to administration of the test substance were not observed in any of the administration groups.

The body weights and amount of weight gain in the maternal animals did not exhibit significantly low values that correlated to the dose in either the 20 or the 40 mg/kg groups throughout the administration period. For food consumption, significantly low values were noted throughout the administration period in the 40 mg/kg group.

Examinations of the ovaries and uterus during the necropsy on day 20 of gestation revealed an impact on only the 40 mg/kg group: specifically, significantly low values for gravid uterine weight, significant increases in the number and rate of fetal mortalities, a significant reduction in the number and rate of surviving fetuses and significantly low values for fetal and placenta weights for both sexes. 

During the external examinations of the surviving fetuses, there was an increase in the rate of occurrence of short phalanges and short tails that correlated to the dose in the 20 and 40 mg/kg groups. Abnormalities of the skeletal frame included missing, damaged and fused tails, while positional abnormalities included missing, damaged and fused bones of the phalanges, with an increase in the rate of occurrence correlating to the doses in the 20 and 40 mg/kg groups. In the 40 mg/kg group, there was also fusion of the metacarpal/metatarsal and phalanx, and missing, damaged and fused metacarpal, and shortening of the tibia and fibula noted in 1~4 animals. On the other hand, there were no increases in abnormalities of the internal organs or tissues of the fetuses that related to administration of the test substance noted in any of the administration groups.

Relative to the fetal ossification, in the 40 mg/kg group, significantly low values were noted in the number of animals with ossification of the sacral and coccygeal vertebrae, thoracic centrum, and metacarpals and metatarsals.

 

From the abovementioned results and under the conditions of this study, the no observable adverse effect level of N, N’-bis (2-methylphenyl) guanidine on maternal and fetal rats is 10 mg/kg/day. On the other hand, clear toxicity was noted in maternal rats with doses of 20 and 40 mg/kg/day, which are believed to be doses that induce abnormalities of the four limbs and tails of fetuses. Additionally, the dose of 40 mg/kg/day is believed to be a dose at which there is an increase in the mortality rate of both maternal animals and fetuses.